posttranslation modification in archaea: lessons from haloferax
TRANSCRIPT
R EV I EW AR T I C L E
Post-translation modification in Archaea: lessons fromHaloferax volcanii and other haloarchaea
Jerry Eichler1 & Julie Maupin-Furlow2,3
1Department of Life Sciences, Ben Gurion University, Beersheva, Israel; 2Department of Microbiology and Cell Science, University of Florida,
Gainesville, FL, USA; and 3Genetics Institute, University of Florida, Gainesville, FL, USA
Correspondence: Jerry Eichler, Department
of Life Sciences, Ben Gurion University,
Beersheva 84105, Israel.
Tel.:+972 8646 1343; fax: +972 8647 9175;
e-mail: [email protected]
Received 12 July 2012; revised 13 November
2012; accepted 13 November 2012. Final
version published online 20 December 2012.
DOI: 10.1111/1574-6976.12012
Editor: Mecky Pohlschroder
Keywords
Archaea; haloarchaea; Haloferax volcanii;
post-translational modification; protein
degradation; proteome.
Abstract
As an ever-growing number of genome sequences appear, it is becoming
increasingly clear that factors other than genome sequence impart complexity
to the proteome. Of the various sources of proteomic variability, post-
translational modifications (PTMs) most greatly serve to expand the variety of
proteins found in the cell. Likewise, modulating the rates at which different
proteins are degraded also results in a constantly changing cellular protein pro-
file. While both strategies for generating proteomic diversity are adopted by
organisms across evolution, the responsible pathways and enzymes in Archaea
are often less well described than are their eukaryotic and bacterial counter-
parts. Studies on halophilic archaea, in particular Haloferax volcanii, originally
isolated from the Dead Sea, are helping to fill the void. In this review, recent
developments concerning PTMs and protein degradation in the haloarchaea
are discussed.
Introduction
The ability to address microorganisms and other life
forms at the level of the genome has revolutionized bio-
logical research. At the same time, it is becoming increas-
ingly clear that a major proprortion of the diversity that
exists within a cell is generated at the level of the prote-
ome. Numerous factors are responsible for the proteome
assuming additional levels of complexity not predicted at
the genome level. These include alternative RNA splicing,
differential expression of a given protein in response to
environmental cues or as a function of developmental
stage, and the plethora of possible protein–protein inter-
actions, as well as post-translational modifications
(PTMs) and regulated protein degradation.
Proteins can be modified post-translationally by the
permanent or temporary covalent attachment of one or
more of several classes of molecules, including sugars, lip-
ids, or other chemical groups. Flexibility in the spatial dis-
tribution of such linked moieties on a target protein and in
the timing of their addition, together with the ability to
introduce changes in the molecular composition of the
bound modifying groups, offers further sources of protein
variability. Intra-molecular disulphide linkages, formed via
the covalent bonding of Cys residues pairs, influence the
three-dimensional conformation of a protein. Disulphide
bonds formed between different protein subunits can yield
multimeric complexes (Fass, 2011). Proteolytic processing
likewise can allow for control of the folding and function
of a target protein. For instance, the post-translational
removal of targeting sequences and inteins respectively per-
mits the cell to control the site where a protein ultimately
resides, as well as the timing and manner in which a pro-
tein can act (Paulus, 2000; Gogarten et al., 2002; Jarvis &
Robinson, 2004; Hegde & Bernstein, 2006). Specifically,
any of these PTMs, either alone or in combination, can
affect protein function, interaction of the modified protein
with binding partners, protein localization, or the rate at
which a protein is degraded, among other traits.
At the same time, regulated protein degradation is crit-
ical for maintaining protein quality and controlling cell
functions. Proteases, which can discern and specifically
degrade proteins compromised by denaturation, misin-
corporation of amino acids, and other damaging events,
are important for regulating cellular homeostasis. Like-
wise, regulated protein degradation processes, in which
FEMS Microbiol Rev 37 (2013) 583–606 ª 2012 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved
MIC
ROBI
OLO
GY
REV
IEW
S
the protease destroys key proteins that may be properly
folded but must be removed at specific times or moved
to locations to enable molecular mechanisms to occur,
are also important to cell function. Central to regulated
protein degradation are proteases that are coupled to
ATP hydrolysis (Gottesman, 2003). These energy-depen-
dent proteases have a self-compartmentalized structure, in
which the proteolytic active sites are housed within a pro-
tein nanoparticle that is chambered, gated, and linked to
an ATPase. The ATPase component of such proteases is
required for the unfolding of protein substrate, opening
of the gate, and facilitating the degradation process
(Lupas et al., 1997; Maupin-Furlow, 2012).
It is now clear that PTMs and regulated protein degra-
dation transpire in all three domains of life, namely Euk-
arya, Bacteria, and Archaea. Still, current understanding
of the archaeal versions of these processes lags behind
that of their eukaryal and bacterial counterparts. In the
following, we review what is known of PTM and regu-
lated protein degradation in the halophilic archaea, lar-
gely focusing on Haloferax volcanii. With the availability
of a complete genome sequence (Hartman et al., 2010),
simple growth requirements and advanced genetic, molec-
ular biology, proteomic and biochemical tools and tech-
niques (DasSarma & Fleishmann, 1995; Allers et al., 2004,
2010; Soppa, 2006; Kirkland et al., 2008b; Dyall-Smith,
2009), H. volcanii represents a strain of choice for molec-
ular studies in Archaea and has provided considerable
insight into the archaeal versions of these protein process-
ing events.
N-glycosylation
N-glycosylation, the covalent linkage of glycan moieties to
select Asn residues of a target protein, was among the
first haloarchaeal PTMs to be described. The Halobacteri-
um salinarum surface (S)-layer glycoprotein was the first
noneukaryotic protein shown to be N-glycosylated
(Mescher & Strominger, 1976). Two different Asn-linked
oligosaccharides modify the S-layer glycoprotein, namely
a repeating sulfated pentasaccharide linked via
N-acetylgalactosamine to Asn-2 and a sulfated glycan
linked by a glucose residue to ten other Asn residues
(Mescher & Strominger, 1978; Lechner et al., 1985a;
Lechner and Wieland, 1989; Wieland et al., 1980, 1983).
The H. salinarum flagellin [since renamed archaellin
(Jarrell & Albers, 2012)] was shown to bear the same gly-
can-linked sulfated polysaccharide (Wieland et al., 1985).
Although applying genetics to identify components of the
H. salinarum N-glycosylation pathway was not possible at
the time, biochemical approaches served to reveal various
aspect of the N-glycosylation pathway of this haloar-
chaeon. As such, it was shown that dolichol phosphate
(DolP) serves as the lipid carrier of the glucose-linked
sulfated glycan decorating 10 sites of N-glycosylation,
whereas dolichol pyrophosphate (DolPP) bears the
repeating sulfated pentasaccharide N-acetylgalactosamine-
linked to S-layer glycoprotein Asn-2 (Wieland et al.,
1980; Lechner and Wieland, 1989). A link between these
phosphodolichol-charged glycans to N-glycosylation was
supported by the observation that in H. salinarum, the
glycan moiety of the DolP-bound sulfated polysaccharide
is also detected on the S-layer glycoprotein and archael-
lins in this species (Lechner et al., 1985a; Wieland et al.,
1985). Moreover, the sulfated polysaccharide is methylat-
ed in the DolP-linked form but not when protein-bound
(Lechner et al., 1985b). The significance of this obser-
vation remains unclear. Finally, studies showing the
ability of H. salinarum cells to modify cell-impermeable,
sequon-bearing hexapeptides with sulfated oligosaccha-
rides localized the N-glycosylation event to the external
cell surface (Lechner et al., 1985a).
Despite these advances, the process of N-glycosylation
in the haloarchaea is currently best understood in H. vol-
canii. The S-layer glycoprotein, comprising the sole com-
ponent of the S-layer, contains seven putative N-
glycosylation sites, namely the motif Asn-X-Ser/Thr, where
X is any residue but Pro. Early studies reported modifica-
tion of the S-layer glycoprotein Asn-13 and Asn-498
positions by a linear string of glucose residues, whereas
Asn-274 and/or Asn-279 were supposedly decorated by a
glycan containing glucose, galactose, and idose (Sumper
et al., 1990; Mengele & Sumper, 1992). More recently, evi-
dence for N-glycosylation of the H. volcanii archaellins,
FlgA1 and FlgA2, was presented (Tripepi et al., 2010,
2012). Likewise, currently unidentified H. volcanii glyco-
proteins of 150, 105, 98, 58, 56, 54, and 52 kDa have been
detected (Zhu et al., 1995; Eichler, 2000), although some
of these species may correspond to the same polypeptide.
Moreover, it remains to be determined whether these
proteins indeed experience N-glycosylation rather than
O-glycosylation. The same is true of LccA, a glycosylated
laccase secreted by H. volcanii (Uthandi et al., 2010). The
H. volcanii S-layer glycoprotein, containing both N- and
O-linked glycans, thus remains the best-characterized
glycoprotein in this species (Sumper et al., 1990).
In the last few years, substantial progress in decipher-
ing the pathway of S-layer glycoprotein N-glycosylation
has been made, with the identification of a series of
archaeal glycosylation (agl) genes encoding proteins
involved in the assembly and attachment of a pentasac-
charide to select Asn residues of the S-layer glycoprotein.
Acting at the cytoplasmic face of the plasma membrane,
AglJ, AglG, AglI, and AglE sequentially add the first four
pentasaccharide residues (i.e. a hexose, two hexuronic
acids and the methyl ester of a hexuronic acid) onto
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Rev 37 (2013) 583–606Published by John Wiley & Sons Ltd. All rights reserved
584 J. Eichler & J. Maupin-Furlow
a common DolP carrier, while AglD adds the final
pentasaccharide residue, mannose, to a distinct DolP
(Abu-Qarn et al., 2007, 2008b; Yurist-Doutsch et al.,
2008, 2010; Guan et al., 2010; Kaminski et al., 2010; Mag-
idovich et al., 2010). The use of DolP by Archaea as the
lipid carrier upon which the N-linked glycan is assembled
also holds true for eukaryal N-glycosylation (Burda &
Aebi, 1999; Hartley & Imperiali, 2012). In contrast, bacte-
rial N-linked glycans are first assembled on a different
isoprenoid, undecaprenol phosphate (Szymanski & Wren,
2005; Weerapana & Imperiali, 2006). N-glycosylation
roles have also been assigned to AglF, a glucose
-1-phosphate uridyltransferase (Yurist-Doutsch et al.,
2010), AglM, a UDP-glucose dehydrogenase (Yurist-Dou-
tsch et al., 2010) and AglP, a methyltransferase (Magido-
vich et al., 2010). Indeed, AglF and AglM were shown to
act in a sequential and coordinated manner in vitro,
transforming glucose-1-phophosphate into UDP-glucu-
ronic acid (Yurist-Doutsch et al., 2010). In a reaction
requiring the archaeal oligosaccharide transferase, AglB
(Abu-Qarn & Eichler, 2006; Chaban et al., 2006; Igura
et al., 2008), the lipid-linked tetrasaccharide and its pre-
cursors are delivered to select Asn residues of the S-layer
glycoprotein. The final mannose residue is subsequently
transferred from its DolP carrier to the protein-bound
tetrasaccharide (Guan et al., 2010) in a reaction requiring
AglR, a protein that either serves as the DolP-mannose
flippase or contributes to such activity (Kaminski et al.,
2012), and AglS, a DolP-mannose mannosyltransferase
(Cohen-Rosenzweig et al., 2012). Current understanding
of H. volcanii N-glycosylation is depicted in Fig. 1.
Insight gained from N-glycosylation in H. volcanii has
served to elucidate aspects of the parallel process in other
halophilic archaea. It was initially shown that H. volcanii
strains lacking either aglD or aglJ could be functionally
complemented by introduction of rrnAC1873 and
rrnAC0149, the respective homologues of these genes
from Haloarcula marismortui (Calo et al., 2010b, 2011).
Like H. volcanii, the haloarchaeon H. marismortui also
originates from the Dead Sea (Oren et al., 1990). Indeed,
subsequent efforts revealed the S-layer glycoprotein of
both species is decorated with N-linked pentasaccharides
comprising a hexose, two hexuronic acids, a methyl ester
of hexuronic acid, and a mannose (Calo et al., 2011).
Still, differences in the N-glycosylation pathways of these
two haloarchaea exist. While in H. volcanii the N-linked
pentasaccharide is derived from a tetrasaccharide sequen-
tially assembled on a single DolP and a final mannose
residue derived from a distinct DolP carrier (Guan et al.,
2010), a similar pentasaccharide N-linked to the
H. marismortui S-layer glycoprotein is first fully assem-
bled on a single DolP and only then transferred to the
protein target (Calo et al., 2011). The finding that H. vol-
canii N-glycosylation pathway components can be
replaced with homologues from other haloarchaea to
yield N-glycan variants has provided a proof-of-concept
for developing H. volcanii in a glyco-engineering platform
designed to produce tailored glycoproteins (Calo et al.,
2011). Such efforts will also exploit the proven ability of
H. volcanii to N-glycosylate introduced nonnative pro-
teins (Kandiba et al., 2012).
The H. volcanii N-glycosylation pathway, involving
multiple glycan-charged DolP carriers, recalls the parallel
eukaryal process. In higher Eukarya, the first seven
subunits of the 14-meric oligosaccharide assembled in the
endoplasmic reticulum are sequentially added to a com-
mon phosphodolichol carrier, whereas the second set of
seven sugar subunits are derived from single mannose- or
glucose-charged DolP (Burda & Aebi, 1999; Helenius &
Aebi, 2004; Hartley & Imperiali, 2012). The H. volcanii
N-glycosylation pathway further resembles its eukaryal
Fig. 1. N-glycosylation in Haloferax volcanii. The H. volcanii S-layer
glycoprotein, a reporter of N-glycosylation in this species, is modified
at Asn-13 and Asn-83 by a pentasaccharide comprising a hexose, two
hexuronic acids, the methyl ester of a hexuronic acid, and a
mannose. The first four subunits of the pentasaccharide are
sequentially assembled onto a DolP carrier via the activities of the
glycosyltransferases, AglJ, AglG, AglI, and AglE. At the same time,
AglD adds the final pentasaccharide residue, mannose, onto a distinct
DolP. Both charged DolP carriers are reoriented to face the cell
exterior, with AglR thought to serve as the DolP-mannose flippase or
to contribute to such activity. AglB acts to transfer the DolP-bound
tetrasaccharide (and its precursors) to select Asn residues of target
proteins, such as the S-layer glycoprotein. The final mannose subunit
is then transferred to the protein-bound tetrasaccharide. AglF, AglM,
and AglP play various sugar-processing roles in the pathway. In the
figure, DolP is presented as a vertical line, while hexoses are
presented as red circles, hexuronic acids are presented as yellow
squares and mannose is presented as a green circle.
FEMS Microbiol Rev 37 (2013) 583–606 ª 2012 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved
Protein modification in the haloarchaea 585
counterpart when one considers that even in cells lacking
the oligosaccharyltransferase, AglB, where pentasaccha-
ride-modified DolP would be expected to accumulate,
only tetrasaccharide-modified DolP could be detected
(Calo et al., 2011). This observation points to the final
mannose of the N-linked pentasaccharide as being added
to the tetrasaccharide already attached to the S-layer gly-
coprotein. This same general strategy is employed in Euk-
arya, where in the Golgi, additional sugar subunits are
attached to oligosaccharides already N-linked to the target
polypeptide. On the other hand, H. marismortui N-glyco-
sylation is similar to the parallel bacterial process in
which a heptasaccharide is assembled by the sequential
addition of seven soluble nucleotide-activated sugars onto
a common undecaprenol phosphate carrier (Szymanski &
Wren, 2005; Weerapana & Imperiali, 2006; Abu-Qarn
et al., 2008a). Yet, although delivered to the lipid-linked
rather than the protein-bound tetrasaccharide, the termi-
nal mannose subunit of the pentasaccharide N-linked to
the H. marismortui S-layer glycoprotein is derived from a
distinct DolP carrier, as in H. volcanii (Calo et al., 2011).
Although the absence or even the perturbation of
N-glycosylation compromises the ability of H. volcanii to
grow in high salt (Abu-Qarn et al., 2007) and modifies
S-layer stability and architecture (Abu-Qarn et al., 2007),
as well as S-layer resistance to added protease (Yurist-
Doutsch et al., 2008, 2010; Kaminski et al., 2010), cells
lacking AglB, and hence unable to perform N-glycosyla-
tion, are viable (Abu-Qarn et al., 2007). As such, it would
seem that this PTM is not essential for H. volcanii sur-
vival, yet nonetheless is advantageous to H. volcanii in
certain scenarios. Thus, one can hypothesize that H. vol-
canii modifies aspects of N-glycosylation in response to
changing growth conditions. This concept has gained
support from recent studies comparing N-glycosylation of
the S-layer glycoprotein in cells grown in 3.4 or 1.75 M
NaCl-containing medium (Guan et al., 2012). At the
higher salinity, S-layer glycoproteins Asn-13 and Asn-83
were shown to be modified by the pentasaccharide
described above, while DolP was shown to be modified
by the tetrasaccharide comprising the first four pentasac-
charide residues, again as discussed above. However, cells
grown at low salinity contain DolP modified by a distinct
tetrasaccharide comprising a sulfated hexose, two hexoses,
and a rhamnose not seen linked to DolP in cells grown at
high salinity. This is likely the same DolP-bound tetrasac-
charide observed by Kuntz et al. (1997) in H. volcanii
cells grown in 1.25 M NaCl-containing medium. The
same tetrasaccharide modified S-layer glycoprotein Asn-
498 in cells grown in low salt, whereas no glycan deco-
rated this residue in cells grown in the high-salt medium.
At the same time, Asn-13 and Asn-83 were modified by
substantially less pentasaccharide at the low-salt condi-
tions (Fig. 2). Hence, in response to environmental salin-
ity, H. volcanii not only modulates the N-linked glycans
decorating the S-layer glycoprotein but also residues sub-
jected to this PTM.
Finally, it should be noted that studies on the metha-
nogens, Methanococcus voltae and Methanococcus marip-
aludis, and the thermophiles, Sulfolobus acidocaldarius,
Pyrococcus furiosus, and Archaeoglobus fulgidus, have also
provided insight into archaeal N-glycosylation (Chaban
et al., 2006; Igura et al., 2008; VanDyke et al., 2009;
Meyer et al., 2011; Jones et al., 2012; Matsumoto et al.,
2012).
Phosphorylation
Phosphorylation is widely appreciated as a covalent form
of PTM that occurs at His, Asp, Ser, Thr, or Tyr residues.
Phosphorylation is rapid, reversible and generates confor-
mational changes in protein structure that mediate an
array of biological responses from signal transduction to
metabolism (Johnson & Barford, 1993). While early stud-
ies suggested that Archaea (and Bacteria) use mainly two-
component systems of His/Asp phosphorylation, it is now
appreciated that Archaea (and Bacteria) also perform Ser/
Thr/Tyr phosphorylation (once thought to be restricted to
eukaryotes) for creating highly sophisticated regulatory
Fig. 2. The Haloferax volcanii S-layer glycoprotein undergoes
differential N-glycosylation as a function of environmental salinity.
Mass spectrometry was used to reveal that when H. volcanii cells are
grown in 3.4 M NaCl-containing medium, Asn-13 and Asn-83 are
modified by the pentasaccharide portrayed in Fig. 1. In the
conditions, Asn-370 and Asn-498 are not modified. When, however,
the cells are grown at lower salt concentrations (i.e. in medium
containing 1.75 M NaCl), S-layer glycoprotein Asn-498 is modified by
a ‘low-salt’ tetrasaccharide comprising a sulfated hexose, two
hexoses, and a rhamnose. At the same time, Asn-13 and Asn-83 are
still modified by the pentasaccharide described above, albeit much
less so. Asn-370 is still not modified. The N-glycosylation status of
Asn-274, Asn-279, and Asn-732 was not considered. In the figure,
hexoses are presented as red circles, hexuronic acids are presented as
yellow squares, mannose is presented as a green circle, and rhamnose
is presented as a blue circle. Positions where no glycosylation is seen
are indicated by ‘9’.
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Rev 37 (2013) 583–606Published by John Wiley & Sons Ltd. All rights reserved
586 J. Eichler & J. Maupin-Furlow
networks (Macek et al., 2008). A number of reviews and
genomic surveys are available that highlight the phosphor-
ylation of archaeal proteins and the enzymes (protein kin-
ases/phosphatases) likely to mediate and/or regulate this
PTM (Leonard et al., 1998; Kennelly & Potts, 1999; Ken-
nelly, 2003; Eichler & Adams, 2005; Tyagi et al., 2010). In
addition, a Phosphorylation Site Database is available
online that provides a guide to some of the Ser/Thr/Tyr-
phosphorylated proteins in Archaea (Wurgler-Murphy
et al., 2004). Thus, the discussion below highlights recent
studies on protein phosphorylation in halophilic archaea
and how this type of PTM may control cellular function.
Phospho-site mapping
Historically, halophilic archaea have provided a useful
model system to advance our understanding of how proteins
can be phosphorylated across domains of life. In fact, the
demonstration that proteins of H. salinarum were reversibly
phosphorylated by a light-regulated retinal-dependent
mechanism was the first report that proteins in Archaea
could be phosphorylated (Spudich & Stoeckenius, 1980).
Later use of molecular biology tools revealed a two-compo-
nent His/Asp phosphorelay system (analogous to the CheA/
CheY system of Bacteria) that was responsible for modulat-
ing the response of H. salinarum to chemotactic and photo-
tactic stimuli (Rudolph & Oesterhelt, 1995; Rudolph et al.,
1995) (Fig. 3). Early study of H. volcanii revealed a Mn2+-
stimulated protein phosphatase activity against synthetic
protein substrates phosphorylated at Ser/Thr residues
(Oxenrider & Kennelly, 1993). More recent studies using
high-throughput methods have facilitated the mapping of
phosphorylation sites on proteins of halophilic archaea (9
phospho-sites in H. volcanii and 81 phospho-sites in H. sali-
narum), thus increasing the number of phospho-sites (from
three total) previously detected in Archaea (Kirkland et al.,
2008a; Aivaliotis et al., 2009). In these high-throughput
approaches, wild-type and mutant strains with enhanced
levels of phosphoproteins [i.e. H. volcanii ΔpanA (protea-
some-activating nucleotidase A) and H. salinarum ΔserB(OE4405R phosphoserine phosphatase)] were used as input
material. Phosphopeptides were enriched from samples by
immobilized metal affinity chromatography and metal oxide
affinity chromatography (in parallel and sequentially), fol-
lowed by tandem mass spectrometry (MS/MS). While a
phospho-site consensus motif was not apparent, the major-
ity of sites mapped to Ser/Thr/Tyr residues.
Protein kinases/phosphatases
Halophilic archaea are predicted to encode histidine pro-
tein kinases and protein phosphatases of the ‘two-compo-
nent’ Asp/His phosphorelay system (Koretke et al., 2000;
Kim & Forst, 2001) (e.g. H. volcanii is predicted to
encode at least 30 histidine protein kinases). The best
studied example of an archaeal Asp/His phospho-relay
system is that of H. salinarum, in which a CheA histidine
protein kinase undergoes ATP-dependent autophosphory-
lation of a His residue and transfers the phosphoryl
group to an Asp residue on CheY, a response regulator
thought to be dephosphorylated by the protein phospha-
tase, CheC (and not CheZ) (Rudolph & Oesterhelt, 1995;
Rudolph et al., 1995; Muff & Ordal, 2007; Streif et al.,
2010). Ultimately, the phosphorylation status of CheY
impacts the ability of this protein to switch the flagellar
motor and regulate cellular movement toward favorable
light and nutrients (Nutsch et al., 2005). Interestingly,
variants of ‘two-component’ histidine protein kinases can
act as Ser/Thr/Tyr protein kinases in eukaryotes (Harris
et al., 1995) and Bacteria (Min et al., 1993; Yang et al.,
1996; Shi et al., 1999; Wu et al., 1999). Whether or not
this alternative type of phosphorylation also occurs in
Archaea is yet unknown.
Many halophilic archaea (including H. volcanii) as well
as the crenarchaeon Thermofilum pendens harbor homo-
logs of the phosphoenolpyruvate phosphotransferase sys-
tem (PTS) (Hartman et al., 2010) (Fig. 4). In analogy to
what is known for Bacteria (Barabote & Saier, 2005), the
H. volcanii PTS is predicted to mediate transfer of
the phosphoryl group on PEP to imported sugars (e.g.
fructose, galacticol) or endogenous dihydroxyacetone via
dihydroxyacetone kinase (Hartman et al., 2010). Halofe-
rax volcanii PTS homologs include a single enzyme I
(PtsI; His~P) and multiple copies of histidine protein
(HPr; His~P), enzyme IIA (EIIA; His~P), enzyme IIB (EIIB;
Cys~P), and enzyme IIC (EIIC; His~P), with the amino acid
PP
P
Htr CheW
CheR
CheB
Flagellarmotor
+ CH3CheY
CheA
–CH3
SAM
Flagella
cw
ccw
Signal
SR
Fig. 3. Protein modification in Halobacterium salinarum taxis. Htrs
are soluble or membrane-bound complexes that associate with signal
receptors (SRs). Htrs signal to a two-component regulatory system
composed of an autophosphorylating histidine kinase CheA, which
mediates phosphotransfer to CheY, the response regulator of the
system. CheY targets the flagellar motor and regulates the switch for
flagellar rotation [clockwise (CW) vs. counterclockwise (CCW)].
Adaptation is promoted by the methylation status of conserved Glu
and Gln residues of Htr, where CheB deamidates Htr Gln residues
prior to O-methylesterification. Htr is methylated by CheR (+CH3) and
demethylated by CheB (�CH3). CheA-mediated phosphorylation
regulates the demethylation activity of CheB.
FEMS Microbiol Rev 37 (2013) 583–606 ª 2012 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved
Protein modification in the haloarchaea 587
residue predicted to be phosphorylated during group
translocation provided in parenthesis. Recent work dem-
onstrates that the PTS gene cluster HVO_1495 to
HVO_1499, encoding PtsI, EIIB, HPr, EIIA, and EIIC
homologs, was highly upregulated as a cotranscript dur-
ing growth on fructose (Pickl et al., 2012). Deletion of
HVO_1499, encoding a homolog of the fructose-specific
membrane component EIIC of this cluster, resulted in
loss of growth on fructose compared to glucose (Pickl
et al., 2012). Thus, the PTS system has a functional
involvement in the metabolism of fructose in H. volcanii.
Atypical RIO-type Ser/Thr protein kinase homologs of
the type 1, type 2, and Bud32 (piD261) families are com-
mon among the Archaea (including the haloarchaea)
(Leonard et al., 1998; Shi et al., 1998; Ponting et al.,
1999; LaRonde-LeBlanc & Wlodawer, 2005a, b; Tyagi
et al., 2010). Protein kinases of the RIO-type 1 family are
distinguished by an STGKEA consensus sequence in their
N-terminal domain and a second region of homology
(IDXXQ, where X represents any amino acid residue) in
their C-terminal domain. Kinases of the RIO-type 2
family often have an N-terminal helix-turn-helix motif
followed by GXGKES and C-terminal IDFPQ sequences.
Members of the Bud32 family of Ser/Thr protein kinases
are associated with the KEOPS (ECK) complex, com-
posed of three additional subunits (Kae1, Pcc1, and
Cgi121), and shown to be required for formation of the
tRNA modification threonylcarbamoyladenosine (t6A) in
yeast (Srinivasan et al., 2011).
A RIO-type 1 homolog (Rio1p, HVO_0135) of H. vol-
canii has been characterized at the biochemical level.
Rio1p purifies as a monomer and can transfer the
c-phosphoryl group of ATP to a1, a protein that forms
the outer rings of 20S proteasomes in H. volcanii
(Humbard et al., 2010b). Rio1p-mediated phosphotrans-
fer is not observed and/or is diminished for a1 variants
T158A, S58A, and T147A. Thus, Rio1p can phosphorylate
a substrate protein (a1) at Ser/Thr residues based on
in vitro assay.
Homologs of the four subunits of the eukaryotic
KEOPS complex are conserved in Archaea, with
H. volcanii-encoding homologs of Pcc1 (HVO_0652) and
Cgi121 (HVO_0013) and a fusion of the Bud32 Ser/Thr
protein kinase to Kae1 (HVO_1895). The gene encoding
the Bud32-Kae1 homolog and its subdomains appear
essential in H. volcanii (Naor et al., 2012), suggesting
Bud32-mediated phosphorylation of Ser/Thr residues is
important for cell function. Homologs of KEOPS (Bud32,
Kae1, Cgi121 and Pcc1) from related Euryarchaeota
(Methanocaldococcus jannaschii and P. furiosus) have been
used for reconstitution of the complex, structural analysis
at the atomic level, heterologous complementation, and
in vitro phosphorylation assays (Hecker et al., 2008; Mao
et al., 2008). From this work, Bud32 is suggested to phos-
phorylate a Thr residue of an insert within the catalytic
cleft of Kae1 and, thus, regulate KEOPS function.
Whether or not Bud32 phosphorylates other proteins in
Archaea besides Kae1 is not clear. Both yeast Bud32 and
its human ortholog, p53-related protein kinase (PRPK),
can phosphorylate p53 (Abe et al., 2001; Facchin et al.,
2003). Likewise, Sulfolobus solfataricus SsoPK5 (a Bud32
homolog) catalyzes the phosphorylation of various
proteins in vitro (Haile & Kennelly, 2011).
In addition to the atypical RIO-type Ser/Thr/Tyr pro-
tein kinases, homologs of the Bacillus subtilis PrkA (Uni-
Prot P39134) are common among halophilic archaea
(e.g. H. volcanii HVO_2849 and HVO_2848). Members
of the PrkA family possess a Walker A-motif of nucleo-
tide-binding proteins and exhibit distant homology to
eukaryotic protein kinases (Fischer et al., 1996). In addi-
tion, amino acid residues within the active site of cyclic
adenosine 3′, 5′-monophosphate (cAMP)-dependent pro-
tein kinase are also conserved in PrkA (Fischer et al.,
1996). B. subtilis PrkA can phosphorylate a 60-kDa pro-
tein at a Ser residue (Fischer et al., 1996). However, fur-
ther analysis is needed to determine the identity of this
protein substrate and confirm that B. subtilis PrkA, and
its haloarchaeal relatives are Ser/Thr protein kinases.
Phosphorylation of proteasomes
Proteasomes are self-compartmentalized proteases,
which undergo a substantial number of post-/co-transla-
tional modifications, including phosphorylation. The
P~HPr3
outin
EIIC1,2EIIB1,2
Sugar
CM
DhaM(EIIA)
DhaL
DhaK
DHA DHAP
P~HPr1,2,3PEP EI
EI~Ppyruvate HPr1,2,3
EIIA1,2
~PEIIA1,2
Sugar -P
HPr3
P~
P~
P~
Fig. 4. PTS of Haloferax volcanii. A schematic diagram of the
H. volcanii phosphotransferase (PTS) system predicted to be
responsible for responsible for the simultaneous transport and
phosphorylation of sugar substrates (e.g. fructose and galacticol) and
for the generation of dihydroyacetone phosphate (DHAP) from
dihydroxyacetone (DHA) by DHA kinase. A series of enzyme
intermediates, including EI, HPr, EIIA, EIIB, EIIC, and DHA kinase
(DhaM, L, K), are predicted to be phosphorylated.
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Rev 37 (2013) 583–606Published by John Wiley & Sons Ltd. All rights reserved
588 J. Eichler & J. Maupin-Furlow
phosphorylation of these complexes is of interest, because
proteasomes are important for cell function (e.g. growth
of H. volcanii) (Zhou et al., 2008). Proteasomes are com-
posed of a 20S catalytic core particle (of a- and b-typesubunits) and regulatory particles, including AAA+ ATP-
ases (homologs of Cdc48/VAT/p97 and Rpt subunits
termed proteasome-associated nucleotidases or PANs)
that mediate energy-dependent protein degradation (Bart-
helme & Sauer, 2012; Maupin-Furlow, 2012). In H. volca-
nii, proteasomes are modified by phosphorylation in
addition to Na-acetylation, methyl-esterification, and
cleavage of b subunit precursors that expose the
N-terminal threonine residue forming the active sites of
20S proteasomes (Table 1) (Wilson et al., 1999; Humbard
et al., 2006, 2010b). Eukaryotic 20S proteasomes and associ-
ated AAA+ ATPases (homologs of PAN termed Rpt1-6 and
Cdc48/VAT/p97) are also altered by phosphorylation and
other forms of covalent modification, such as the attach-
ment of O-linked N-acetylglucosamine, Ne- and Na-acety-lation, N-myristoylation, and cleavage of b subunit
precursors (Zhang et al., 2007; Ewens et al., 2010).
Haloferax volcanii 20S proteasomes and associated
PANs are phosphorylated at Ser and Thr residues. To
facilitate phospho-site mapping, 20S proteasomes and
PAN proteins were purified from H. volcanii by tandem
affinity chromatography using His6- and StrepII-tags
(Humbard et al., 2006, 2010b). Sites of phosphorylation
were identified by MS/MS (with precursor ion scanning)
and included b Ser129, a1 Thr147, and a2 Thr13/Ser14
(not distinguished) of 20S proteasomes and Ser340 of
PAN-A (Humbard et al., 2006, 2010b). A phospho-site
for PAN-B was not identified (Humbard et al., 2010b).
MS/MS analysis of phosphopeptides enriched from
H. volcanii proteomes suggests Cdc48/VAT/p97 homologs
are also phosphorylated; however, sites of modification
were not identified by this high-throughput method
(Kirkland et al., 2008a). In eukaryotes, phosphorylation
and acetylation regulate the function of Cdc48/VAT/p97
proteins.
Phosphorylation of the a-type subunits of H. volcanii
proteasomes has been investigated at various stages of cell
growth and assembly states. Humbard et al. (2010b) sepa-
rated the a-type subunits (of cell lysate and 20S proteo-
somes purified by affinity chromatography with bsubunits) by two-dimensional gel electrophoresis (2DE)
and detected a1 and a2 by immunoblot using polyclonal
antibodies specific to each protein. Phosphorylated iso-
forms (two specific for a1 and one specific for a2) were
determined by shifting the 2DE-protein spot to a more
basic pI after removal of the acidic phosphate groups by
phosphatase treatment. Of the two phosphorylated iso-
forms of a1 detected, the most acidic form was found
throughout growth, assembled in 20S proteasomes. In
contrast, the least phosphorylated isoform of a1 was pres-
ent as both unassembled and assembled subunits of 20S
proteasomes and was detected at reduced levels in later
stages of growth (Humbard et al., 2010b). The phosphor-
ylated isoform of a2 was also found associated with 20S
proteasomes. Thus, phosphorylation of the a-type pro-
teins is suggested to influence their assembly and/or be
important for 20S proteasome function.
Site-directed mutagenesis has been used to determine
the biological role of proteasome phosphorylation in
H. volcanii. Haloferax volcanii strains expressing a1 pro-
teins with Ala modifications in Ser/Thr residues likely to
be phosphorylated (based on MS analysis and an inability
to accept a phosphoryl group from Rio1p, in vitro) dis-
play dominant negative phenotypes for cell viability and
colony color (i.e. white vs. red) (Humbard et al., 2010b).
Thus, phosphorylation of the a1 subunit of 20S protea-
somes appears to be closely linked to cell growth and pig-
mentation (i.e. production of carotenoids) (Humbard
et al., 2010b). Interestingly, H. volcanii proteasomal
mutant strains deficient in the synthesis of PAN-A display
a striking increase in the number of cellular proteins that
are phosphorylated, suggesting an added link between
protein phosphorylation and proteasome function (e.g.
phosphorylation may target proteins for destruction by
energy-dependent proteases, in analogy to eukaryotes and
Bacteria) (Kirkland et al., 2008a).
Protein acetylation
Protein acetylation is the covalent attachment of an acetyl
group to a protein. In general, acetylation can impact
protein function, stability, and interactions with other
molecules. Two types of protein acetylation are known to
occur in living cells, Na-acetylation and Ne (or lysine)-
acetylation, with high-energy molecules, such as acetyl-
CoA, providing the acetyl group for these modifications.
Na-Acetylation is an irreversible mechanism in which an
acetyl group is covalently attached to the a-amino group
Table 1. PTMs of Haloferax volcanii proteasomes
Subunit Phosphorylated
Methyl-
esterified
Na-
acetylated
Exposed by
autocleavage
a1 Thr147 Asp20, Glu27,
Glu62, Glu112,
Glu161
Met1 n.d.
a2 Thr13/Ser14 n.d. Met1 n.d.
b Ser129 n.d. n.d. Thr50
PAN-A Ser340 n.d. n.d. n.d.
Residue number according to protein sequence in GenBank [GI:30
0669661 (a1), GI:12229945 (a2), GI:292655712 (b), GI:302425218
(PAN-A)].
FEMS Microbiol Rev 37 (2013) 583–606 ª 2012 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved
Protein modification in the haloarchaea 589
of the N-terminal amino acid of a protein. In contrast,
Ne-acetylation occurs when the e-amino group of a lysine
residue is reversibly modified by the covalent attachment
of an acetyl group. Studies on H. volcanii have furthered
our understanding of both processes in Archaea.
Na-acetylation
Our current understanding of the prevalence of Na-acety-lation in Archaea is largely based on MS-based proteomic
surveys (Soppa, 2010; Maupin-Furlow et al., 2012). Like
Bacteria, Na-acetylation of ribosomal proteins appears
common among Archaea (Kimura et al., 1989; Hatakey-
ama & Hatakeyama, 1990; Klussmann et al., 1993; Mar-
quez et al., 2011). Interestingly, the number of proteins
reported to be Na-acetylated varies greatly among the dif-
ferent archaeal groups. In haloarchaea (apparently unlike
Bacteria), a relatively high proportion (14–29%) of the
proteome is modified by Na-acetylation (i.e. H. salina-
rum, Natronomonas pharaonis, and H. volcanii) (Falb
et al., 2006; Aivaliotis et al., 2007; Kirkland et al., 2008b).
Likewise, Na-acetylation appears to impact a large per-
centage of the S. solfataricus proteome, based on the find-
ing of 17 Na-acetylated N-termini of 26 total detected by
MS (Mackay et al., 2007). Interestingly, to date, only a
single protein (the a subunit of the 20S proteasome) is
reported to be Na-acetylated among the methanogens,
suggesting that this form of modification may be rare in
this group of Archaea (Forbes et al., 2004; Zhu et al.,
2004; Enoki et al., 2011).
In H. volcanii, like other Archaea, many of the
Na-acetylated proteins appear to be generated by a NatA-
type activity [i.e. acetylation of penultimate Ser or Ala
residues exposed after removal of N-terminal methionine
residues by methionine aminopeptidase (MAP)] (Fig. 5).
Na-Acetylated N-terminal methionine residues with pen-
ultimate Asp and Asn residues are also detected, consis-
tent with the NatB-like activity of eukaryotes (Kirkland
et al., 2008b). Proteins with a Na-acetylated N-terminal
methionine residue followed by a small penultimate resi-
due (Ser, Ala, Thr, Pro, and Gly) are also detected, sug-
gesting that Na-acetylation can restrict their cleavage by
MAP. Interestingly, many of the proteins of halophilic ar-
chaea (including those of H. volcanii) that are Na-acety-lated are also readily identified by semi-quantitative MS
spectral counting in unmodified and/or MAP-cleaved
forms (Falb et al., 2006; Aivaliotis et al., 2007; Kirkland
et al., 2008b). Liquid chromatography-multiple reaction
monitoring (LC-MRM) MS, a technique that provides a
more accurate perspective on the abundance of the
Na-acetylated state of a protein, reveals that the a1proteins that form 20S proteasomes are primarily in an
Na-acetylated Met form, as compared to the MAP
cleaved form (103 : 1 ratio), in H. volcanii (Humbard
et al., 2009). Whether or not Na-acetylation efficiency is
also near 100% for other H. volcanii protein substrates
and/or can be altered by growth conditions remains to be
determined.
While proteins with Na-acetylated Met-Gln and Met-
Asn sequences are not prevalent in Archaea, 20S protea-
some a-type subunits with these N-terminal sequences are
specifically Na-acetylated in the Euryarchaeota, H. salina-
rum, N. pharaonis, H. volcanii, and Methanothermobacter
thermoautotrophicus (Falb et al., 2006; Humbard et al.,
2006; Aivaliotis et al., 2007; Enoki et al., 2011). Site-direc-
ted mutagenesis of the N-terminal Met-Gln sequence of
the H. volcanii 20S proteasome a1 protein has been used
to further investigate this specific Na-acetylation (Hum-
bard et al., 2009). Variants of a1 were expressed in vivo
and analyzed by MS to detect alterations in N-terminal
modification (i.e. Na-acetylation, MAP cleavage). A Q2A
substitution rendered the a1 protein susceptible to cleav-
age by MAP followed by Na-acetylation of the penulti-
mate Ala by an apparent NatA-type activity similarly to
most Na-acetylated proteins in haloarchaea. However,
the N-termini of a1 proteins with the small penultimate
amino acid residues Ser and Val were detected
A
S
GV
P
T
D ENI W FL K Q
MAP cleaved (n = 143)
A
S
M
V TKD
F QLNα-acetylated (n = 72)
MS
MAMT
MPMGMV
MNMD MQ
Fig. 5. Haloferax volcanii N-terminal proteome modified by
Na-acetylation (left) and/or cleavage by MAP (right). Pie charts based
on the N-terminal proteome of H. volcanii that was detected by MS/
MS (Kirkland et al., 2008b). Based on this proteomic analysis,
H. volcanii proteins are often cleaved by MAP and/or Na-acetylated.
Penultimate residues exposed by MAP are often small and uncharged
(Gly, Ala, Pro, Val, Ser, or Thr). Of the N-termini that are acetylated, a
relatively equal divide exists between proteins Na-acetylated at their
N-terminal Met vs. a residue exposed after MAP cleavage (with
Na-acetylation of exposed Ser and Ala residues common). Among
proteins with Na-acetylated N-terminal Met residues, most (over
80%) have the Met residue followed by a small, uncharged residue
(Gly, Ala, Pro, Val, Ser, or Thr) typically cleaved by MAP.
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Rev 37 (2013) 583–606Published by John Wiley & Sons Ltd. All rights reserved
590 J. Eichler & J. Maupin-Furlow
predominantly in the uncleaved forms, with
Na-acetylated methionine residues intact. Alteration of
penultimate amino acid residues to Asp, Pro, and Thr
resulted in a mixture of a1 protein in the Na-acetylatedmethionine, MAP cleaved and/or unmodified forms. Thus,
the enzyme(s) responsible for Na-acetylation of the a1N-terminal Met appears to have relaxed sequence specific-
ity with regard to the penultimate residue of the substrate.
Furthermore, only the Q2A substitution rendered the
N-terminus of a1 fully susceptible to MAP cleavage, sug-
gesting that structural elements of a1 or interacting part-
ners/chaperones may mask primary N-terminal sequences
that are otherwise optimal for MAP cleavage (e.g. Met-Ser
of the a1 Q2S variant). Interestingly, Na-acetylation of a1Met appears important in gating the 20S proteasome,
based on the enhanced peptidase activity of 20S protea-
somes with the MAP-cleaved a1 Q2A variant and the
inability of the gene encoding the a1 Q2A variant to com-
plement the a1 mutation for growth at ‘low’ salt
(� 1.3 M NaCl). Insight into archaeal enzyme(s) that
mediate Na-acetylation is provided through comparative
genomics, in vitro reconstitution of Na-acetyltransferaseactivity and crystallography. Members of the GNAT super-
family that use acetyl-CoAs to acylate their cognate
substrates, including the Na-acetylation of proteins, are
widely distributed among Archaea (Vetting et al., 2005).
Sulfolobus solfataricus ssArd1 (SSO0209) is an archaeal
GNAT related to Na-acetyltransferases that has been dem-
onstrated to acetylate the N-terminal residue (Ser) of the
DNA-binding protein, Alba (Mackay et al., 2007). Much
like the eukaryal Ard1 of NatA, SsArd1 preferentially
acetylates N-terminal Ser and Ala residues exposed after
methionine removal (Mackay et al., 2007). SsArd1 also
catalyzes appreciable Na-acetylation of N-terminal
Met-Glu and Met-Leu sequences, similar to Nat3 of NatB
in eukaryotes (Mackay et al., 2007). While ssArd1 can
Na-acetylate a variety of proteins in vitro, it shows prefer-
ence for proteins with N-termini that are disordered in
crystal structures (Mackay et al., 2007). Thus, it is unclear
whether ssArd1 functions post- or co-translationally in the
cell. The archaeal ssArd1 is thought to represent an ances-
tral form of some eukaryal Na-acetyltransferases based on
its relaxed sequence specificity. Recent crystallography of
SsArd1 now provides structural detail for analysis of this
ancestral function (Oke et al., 2010).
Na-acetylation and protein stability
Based on analogy to eukaryotes, Na-acetylation is
predicted to regulate protein turnover in H. volcanii and
other haloarchaea. The long-held argument that
Na-acetylation stabilizes a protein comes from several
lines of indirect evidence (Meinnel et al., 2006). For
example, proteins modified by Na-acetylation are often
over-represented in protein abundance profiles and
comprise a high proportion of the proteome (Falb et al.,
2006; Aivaliotis et al., 2007; Kirkland et al., 2008b;
Martinez et al., 2008). Furthermore, Na-acetylationblocks the Na-amino group of a protein from further
modification by destabilizing processes such as ‘linear’
ubiquitylation (Meinnel et al., 2005). However, recent
evidence suggests Na-acetylation can mark a protein for
destruction by the ubiquitin-proteasome system using a
mechanism named the Ac/N-end rule (Hwang et al.,
2010). The Ac/N-end rule is based on the finding that an
E3 ubiquitin ligase (named Doa10) can recognize proteins
with acetylated N-termini and facilitate their ubiquityla-
tion (at internal lysine residues) and degradation by pro-
teasomes in yeast (Hwang et al., 2010). To rationalize this
discrepancy between Na-acetylation in protein stability
and degradation, Hwang et al. (2010) provide a model
in which nascent proteins can ‘hide’ their acetylated
N-termini by rapid folding, interaction with chaperones,
and/or assembly into appropriate multi-subunit com-
plexes. Such sequestration of N-termini would render the
Na-acetylation-based degradation signals inaccessible for
recognition by the Doa10 E3 ubiquitin ligase, ultimately
stabilizing the protein. In contrast, delayed or defective
protein folding would expose acetylated N-termini and
allow for Doa10-dependent ubiquitylation and proteolysis
by proteasomes.
Insight into a potential archaeal Ac/N-end rule pathway
is provided by study of the a1 protein of 20S protea-
somes in H. volcanii (Humbard et al., 2009; Varshavsky,
2011). Here, the identity of the N-terminal penultimate
residue of a1 was found to dramatically alter the concen-
tration of a1 protein in the cell. In particular, the
levels of N-terminal a1 variants that were partially non-
Na-acetylated were remarkably higher than the levels of
Na-acetylated (wild type) a1 protein. Furthermore, most
of the a1 proteins associated in 20S proteasomes had
acetylated N-termini. Thus, proteasomal partners are
predicted to obstruct recognition of the acetylated
N-terminal domain of a1 and prevent its proteolytic
destruction by an Ac/N-end rule pathway.
Ne-acetylation
The reversible and differential Ne-acetylation of proteins
can have a major impact on transcription, translation,
stress response, detoxification, and carbohydrate and
energy metabolism (Hu et al., 2010; Jones & O’Connor,
2011; Thao & Escalante-Semerena, 2011). In eukaryotes,
histones are well known to be modified by Ne-acetyla-tion, in addition to Na-acetylation, methylation,
phosphorylation, ubiquitylation, ADP ribosylation,
FEMS Microbiol Rev 37 (2013) 583–606 ª 2012 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved
Protein modification in the haloarchaea 591
glycosylation, and sumoylation (Shiio & Eisenman, 2003).
Numerous bacterial proteins are also differentially Ne-acetylated, with ‘K-acetylomes’ (subproteomes composed
of proteins with Ne-acetylated lysine residues) thought to
rival phosphoproteomes (Aka et al., 2011). In contrast,
Ne-acetylation of archaeal proteins is poorly understood.
In Archaea, only a few proteins are known to be
Ne-acetylated. A couple of early studies, focused on deter-
mining the amino acid sequence of 2Fe–2S ferredoxins
from the haloarchaea H. salinarum and H. marismortui,
revealed that a lysine residue near the C-terminus of these
proteins is conserved and Ne-acetylated (Hase et al.,
1978, 1980). Another archaeal protein that is Ne-acety-lated is Alba, a chromatin protein of S. solfataricus. Alba
is not only Na-acetylated on its N-terminal Ser (as dis-
cussed above) but is also Ne-acetylated on lysine 16 (Bell
et al., 2002). Acetylation of Alba Lys16 is mediated by the
protein acetyltransferase, Pat (Marsh et al., 2005). Pat is a
homolog of Salomonella Pat (YfiQ) and a member of the
family of NDP-forming acetyl-CoA synthetase enzymes
with GNAT domains (Starai & Escalante-Semerena,
2004). Alba Lys16 can be deacetylated by an NAD+-
dependent histone deacetylase (HDAC) class III homolog
of S. solfataricus (Sir2) (Bell et al., 2002). Ne-Acetylationof Alba reduces its binding affinity for DNA and RNA
and strongly prevents its ability to inhibit the DNA heli-
case activity of the mini-chromosome maintenance pro-
tein (Bell et al., 2002; Jelinska et al., 2005; Marsh et al.,
2006). Thus, Ne-acetylation of Alba is thought to have a
global impact on chromatin packaging and gene expres-
sion in Crenarchaeota, such as S. solfataricus (Wardle-
worth et al., 2002; Zhao et al., 2003).
Haloferax volcanii has served as a model for under-
standing the importance of histone acetyltransferase
(HAT) and HDAC gene homologs to archaeal cell func-
tion. In particular, gene homologs for three HATs (Pat1,
HVO_1756; Pat2, HVO_1821; Elp3, HVO_2888) and two
HDACs (Sir2, HVO_2194; HdaI, HVO_0522) were tar-
geted for deletion from the H. volcanii genome (Altman-
Price & Mevarech, 2009). Pat1 and Pat2 are related to the
S. solfataricus Pat, Elp3 is related to the yeast Elp3 sub-
unit of the elongator complex possessing acetyltransferase
activity, Sir2 is a class III HDAC homolog similar to
S. solfataricus Sir2, and HdaI is a class II HDAC homolog
related to yeast Hda1. Single deletion of sir2, pat1, pat2,
or elp3 genes or double deletion of pat1 with either pat2
or elp3 was found to have no detectable impact on the
viability of H. volcanii cells. In contrast, hdaI appeared
essential, based on the finding that the gene could only
be deleted when a wild-type copy of hdaI was provided in
trans. Attempts to create an elp3 deletion in any of the
pat2 null strains were unsuccessful, implying that these
two mutations are synthetically lethal and affect a single
function or pathway (Altman-Price & Mevarech, 2009).
Thus, Elp3- and Pat2-mediated acetylation and HdaI-
mediated deacetylation of lysine residues are predicted to
be important in H. volcanii.
Hypusine modification
Hypusine [Ne-(4-amino-2-hydroxybutyl)-L-lysine] is
formed upon PTM of a conserved lysine residue and
is found only in eukaryotic translation ‘initiation’ factor
5A (eIF5A) and the related archaeal aIF5A (Park et al.,
1981, 2010; Schumann & Klink, 1989; Bartig et al., 1992).
Hypusine is essential for the activity of eIF5A/aIF5A
(including that of H. salinarum), now considered impor-
tant in translation elongation (Wagner & Klug, 2007;
Saini et al., 2009; Park et al., 2010). In hypusine modifica-
tion of eIF5A, deoxyhypusine synthase (DHS) transfers a
4-aminobutyl moiety from spermidine to the e-amino
group of the conserved lysine residue to form a dexoxy-
hypusine intermediate, which is hydroxylated to a hypu-
sine residue by deoxyhypusine hydroxylase (DOHH)
(Fig. 6). In Archaea, DHS homologs are widespread,
suggesting the lysine residue of aIF5A is modified to a
deoxyhypusine residue by an enzyme similar to eukaryotic
DHS. In contrast, DOHH is an oxygen-dependent
enzyme, and its homologs are rare in (the often anaerobic)
Archaea. Thus, generation of the hypusine-modified form
of aIF5A from its deoxyhypusine precursor likely involves
a second enzyme distinct from DOHH in Archaea.
Spermidine
NH2
Lys
eIF5A
NH
Lys
eIF5A
H2N
NH
Lys
eIF5A
H2N
–OH
O2 H2O
1,3-diaminopropane
DHS DOHH
Deoxyhypusine Hypusine
donor-H2 acceptor
Fig. 6. Hypusine modification of eIF5A.
Hypusine is a universal modification in Archaea
and eukaryotes on a single type of protein
(eIF5A) by a sequential series of enzyme
reactions. DHS transfers a 4-aminobutyl
moiety from spermidine to the e-amino group
of a specific lysine residue on eIF5A. DOHH
hydroxylates the modified lysine to form
hypusine.
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Rev 37 (2013) 583–606Published by John Wiley & Sons Ltd. All rights reserved
592 J. Eichler & J. Maupin-Furlow
Protein methylation
Protein methylation is a type of PTM that can mediate
many important biological processes, spanning from gene
regulation and signal transduction to protein stability
(Clarke, 1993; Lee et al., 2005; Paik et al., 2007). In this
PTM, nucleophilic oxygen, nitrogen, and sulfur atoms on
the polypeptide chain can be methylated, resulting in the
generation of methyl esters, methyl amines, methyl
amides, and other modifications (with only the hydroxyl
groups of Ser, Thr and Tyr not detected in methylated
forms). Methylation of the side chain hydroxyl group of
dicarboxylic amino acid residues (Asp, Glu) and the
hydroxyl group generated by deamidation of glutamine
residues (O-methylesterification) is typically reversible
and regulated. Recent evidence also supports the revers-
ible methylation of the amino groups in the side chains
of lysine (mono-, di-, and tri-methylated forms) and argi-
nine (mono- and di-methylated forms) residues of pro-
teins (Lee et al., 2005). S-adenosylmethionine (SAM)
commonly serves as the methyl group donor for the
methylation of proteins by methyltransferases that are
often specific for their protein substrate, while methyles-
terases and demethylases can remove the methyl groups.
In haloarchaea, O-methylesterification of proteins has
been detected and is the focus of the discussion below.
O-methylesterification
The methyl-accepting taxis proteins or transducers are a
large group of membrane-associated proteins that are typ-
ically methylated at glutamate residues (some of which
originate from glutamine residues via deamidation) (Por-
ter et al., 2011). Transducer homologs are relatively wide-
spread among Bacteria and Euryarchaeota (including
halophiles, methanogens, thermococci, and others). In
general, transducers act as (or interact with) sensory
receptors for a specific stimulus (e.g., light, attractant)
and are demethylated in response. The methylation state
of the transducer provides a ‘primitive memory’ or adap-
tation to a two-component regulatory system (CheA/
CheY) that switches the flagellar motor and alters cellular
movement. Among the archaeal transducers, the best
studied are the halobacterial transducers (Htrs) of H. sali-
narum, which comprise a group of 18 homologs (6 solu-
ble and 12 membrane-spanning) with 1–3 conserved
methylation sites per protein (Ng et al., 2000; Pfeiffer
et al., 2008) (Fig. 3). Early studies of H. salinarum dem-
onstrate methyl-[3H]-labeling of proteins and volatile
products of demethylation that are specific to taxis
and define the methylated proteins as Htrs (soluble and
membrane-spanning), based on the abolishment of
methyl-[3H]-labelling after gene deletion and site-directed
mutagenesis (Alam et al., 1989; Sundberg et al., 1990;
Nordmann et al., 1994; Lindbeck et al., 1995; Brooun
et al., 1997; Perazzona & Spudich, 1999; Storch et al.,
1999). Recently, MS-based proteomes have been used to
map three methylation sites in two of the six soluble Htrs
and 19 methylation sites in 10 of the 12 predicted mem-
brane-spanning Htrs (Koch et al., 2008). Sites include
singly methylated pairs of Glu and/or Gln, Asp-Glu, and
Ala-Glu residues (with Glu or deamidated Gln residues
methylated). Evidence supports the function of CheB in
deamidation and demethylation of the Htrs and CheR in
transfer of the methyl group from SAM co-factor to con-
served Glu or deamidated Gln residues of Htr (Perazzona
& Spudich, 1999; Koch & Oesterhelt, 2005; Koch et al.,
2008). Selective demethylation of Htr sites (e.g. one site is
demethylated upon stimulation by attractant, and another
site is demethylated upon simulation with repellent) is
proposed to control a diverse array of biological
responses, in analogy to the bacterium Bacillus subtilis
(Streif et al., 2010).
Like transducers, archaeal 20S proteasomes are
modified by O-methylesterification. In particular, five
unique acidic residues (Asp20, Glu27, Glu62, Glu112, and
Glu161) of the H. volcanii 20S proteasome a1 subunit are
methylated (Table 1) (Humbard et al., 2010b), as
detected by MS/MS with methods devoid of added meth-
anol (Chen et al., 2010). O-linked methylesterification is
typically a reversible form of PTM. Furthermore, both
methylated and unmethylated forms of a1 are readily
detected in the cell (Humbard et al., 2010b). Thus,
O-methylesterification of a1 is thought to be regulated
and to modulate proteasomal activity (e.g. stability in low
salt, high temperature, interaction with AAA+ ATPase
partners). While the enzyme responsible for a1 methyla-
tion is as yet unknown, a SAM-dependent methyltransfer-
ase homolog (HVO_1093) co-transcribed with the a1gene is a likely candidate (Gil et al., 2007).
N-terminal methionine removal
Methionine residues can be removed from the N-terminus
of proteins in all domains of life. Archaea and Eukarya use
methionine (Met) for the initiation of protein synthesis
(Ramesh & RajBhandary, 2001), and this N-terminal methi-
onine can be removed by a MAP as the protein emerges
from the ribosome (Lowther & Matthews, 2000). In contrast
to Met, Bacteria, and the related organelles of eukaryotes
(i.e. mitochondria and chloroplasts) use N-formylmethio-
nine (fMet) for initiation of protein synthesis. fMet is a
derivative of Met in which a formyl group has been added
to the amino group and is important for bacterial cell func-
tion (Guillon et al., 1992). With rare exceptions (Milligan &
Koshland, 1990) the N-formyl group is typically removed
FEMS Microbiol Rev 37 (2013) 583–606 ª 2012 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved
Protein modification in the haloarchaea 593
from bacterial proteins by a peptide deformylase (Mazel
et al., 1994). After deformylation, the resulting N-terminal
methionine can be removed (Solbiati et al., 1999).
MAPs (EC 3.4.11.18) are metalloenzymes common to
all domains of life that remove N-terminal leading methi-
onine residues from nascent peptides during the early
stages of protein synthesis. MAPs generally cleave sub-
strates with penultimate residues that are one of the seven
small and uncharged amino acids (i.e. Gly, Ala, Ser, Thr,
Pro, Cys, and Val). MAPs belong to the MEROPS pepti-
dase family, M24 (clan MG), and are divided into two
types (types I and II), with the C-terminal domain of type
II distinguished by insertion of an extra � 60 amino acid
helical subdomain (Arfin et al., 1995). Bacteria typically
only contain the type I MAP (with some exceptions),
Archaea contain only type II MAP, while eukaryotes pos-
sess both type I and type II MAPs (Bazan et al., 1994;
Arfin et al., 1995; Lowther & Matthews, 2002).
Among the archaea, MAPs from the hyperthermophiles
P. furiosus (PfMAP) and Thermococcus sp. NA1 have been
purified in enzymatically active forms (Tsunasawa et al.,
1997; Tahirov et al., 1998; Lee et al., 2006). Of these two
enzymes, PfMAP is the most extensively studied. Similar
to the bacterial and eukaryal MAPs, PfMAP cleaves
N-terminal methionine from substrates whose penulti-
mate amino acid residues are Gly, Ala, Ser, Thr, Pro, and
Val, based on release of N-terminal methionine from the
synthetic peptide, Met-X-Ala-Ala-Ala (where X represents
the 19 common amino acids, excluding Cys) (Tsunasawa
et al., 1997). PfMAP also provided the first crystal struc-
ture of a type II MAP (Tahirov et al., 1998). Those
PfMAP residues (Asp82, Asp94, His153, Glu187, and
Glu280) that coordinate the two catalytic metal ions are
conserved in the Escherichia coli type I MAP crystal struc-
ture (with the identity of the metal ions used for catalysis
still under debate) (Roderick & Matthews, 1993). Like-
wise, the two positively charged residues (His62 and
His161) in the active site cleft of PfMAP that are thought
to shuffle protons from the nuclear center to the solvent
during catalysis are also conserved in E. coli MAP
(Roderick & Matthews, 1993; Tahirov et al., 1998).
While MAPs have not been purified from halophilic
archaea, type II MAP homologs are conserved and large-
scale proteomic studies provide evidence for MAP activity
in this group of microorganisms. In particular, nonredun-
dant N-terminal peptides identified by MS have been reli-
ably mapped to a large number of proteins from
H. salinarum (606 proteins), N. pharaonis (328 proteins),
and H. volcanii (236 proteins) (Falb et al., 2006; Aivaliotis
et al., 2007; Kirkland et al., 2008b). Of these proteins,
most (60–70%) were missing an N-terminal methionine
and presented N-termini suggesting that MAP cleavage
occurred predominantly when the penultimate residue
was small and uncharged (Gly, Ala, Pro, Val, Ser, or Thr)
(for H. volcanii, see Fig. 5). Interestingly, recent compari-
son of large data sets of nonredundant N-terminal pep-
tides detected by MS from diverse organisms suggests
that MAP cleavage efficiency is conserved across all
domains of life (Helbig et al., 2010).
Signal peptide cleavage
Proteins destined to reside beyond the confines of the
cytoplasm (i.e. secretory and membrane proteins)
are often synthesized as preproteins bearing cleavable
N-terminal targeting sequences termed signal peptides. In
H. volcanii, signal peptides direct preproteins to both the
Sec and the Tat translocation systems (Fig. 7) (Pohlsch-
roder et al., 2005; Calo & Eichler, 2011). Although the
major protein species of H. volcanii, the S-layer glycopro-
tein, relies on the Sec translocation system, bioinformat-
ics-based analysis predicts that the Tat translocation
system is preferentially used by exported H. volcanii
proteins (Dilks et al., 2003; Storf et al., 2010). In each sys-
tem, once the signal peptide has served its purpose,
namely targeting a preprotein to a proteinaceous translo-
cation complex, it is cleaved from the mature protein by
the actions of type I signal peptidase (SPase), although, as
considered below, the majority of Tat pathway lipoprotein
substrates are apparently processed by a type II SPase.
While general aspects of archaeal type I SPase biology have
been considered in several recent reviews (Ng et al., 2007;
Jarrell et al., 2010; Calo & Eichler, 2011), this enyzme has
only been biochemically addressed in a limited number of
Archaea to date, including H. volcanii (Fine et al., 2006;
Fink-Lavi & Eichler, 2008). In H. volcanii, genes encoding
two distinct type I SPases, Sec11a (HVO_2603) and
Sec11b (HVO_0002), are found (Fine et al., 2006). Of the
two, HVO_0002 is an essential gene. Observed differences
in the activities of the two purified enzymes toward a sig-
nal peptide-bearing reporter protein suggest that Sec11a
and Sec11b possess distinct substrate preferences.
At the molecular level, H. volcanii type I SPases present
a mosaic of eukaryal, bacterial, and archaeal properties
(Fine et al., 2006; Calo & Eichler, 2011). Like their eukar-
yal counterparts, the H. volcanii enzymes have replaced
the conserved lysine of the serine-lysine catalytic dyad
found in bacterial SPases with a histidine. Site-directed
mutagenesis studies revealed that both of these residues
are essential for Sec11b activity (Fink-Lavi & Eichler,
2008). Likewise, a third H. volcanii Sec11b residue, Asp-
280, was deemed as being essential for catalytic activity of
the enzyme. The equivalent residues were also shown to
be necessary for M. voltae SPase activity (Bardy et al.,
2005). As the yeast type I SPase also requires these resi-
dues for its functionality, it would appear that the
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Rev 37 (2013) 583–606Published by John Wiley & Sons Ltd. All rights reserved
594 J. Eichler & J. Maupin-Furlow
archaeal enzyme relies on a catalytic mechanism like that
employed by its eukaryal counterpart. On the other hand,
yeast SPase also requires the presence of the equivalent of
H. volcanii Sec11b Asp-273 (van Valkenburgh et al.,
1999), a residue not essential for the activity of the halo-
archaeal enzyme (Fink-Lavi & Eichler, 2008). At the same
time, the H. volcanii enzymes also share traits with their
bacterial counterparts. Tagged versions of Sec11a and
Sec11b were purified as single polypeptides, each able to
cleave the signal peptide from a reporter protein, suggest-
ing that the H. volcanii enzymes function independently
of other proteins, as do bacterial SPases. Eukaryal type I
SPases, by contrast, exist as part of a multimeric complex
in which additional proteins apart from Sec11 are essen-
tial for activity (Evans et al., 1986; YaDeau et al., 1991).
Still, the possibility remains that the H. volcanii SPases
operate optimally only when in complex with additional
components not captured under the conditions employed
nor identified in genome searches.
As discussed in more detail below, H. volcanii is
thought to contain a large number of lipoproteins. While
type II SPases remove characteristic lipoprotein signal
sequences, thereby unmasking the site of such lipid-based
PTM in target proteins, no such enzyme has been
detected in H. volcanii or any other archaea. It remains
unclear whether this reflects the absence of such an
archaeal enzyme or whether the archaeal enzyme, possibly
designed to act on the ether-based lipids of the archaeal
membrane, shares little sequence homology with its bac-
terial counterpart and, as such, has been overlooked.
Unlike type I SPases that cleave Sec or Tat transloca-
tion signal peptides, type III SPases process the unique
signal peptides found in archaellins, pili, and other cell-
surface structures (Albers et al., 2003). Analysis of the
H. volcanii genome reveals the presence of several prepro-
teins bearing a type III signal peptide, as well as a single
type III signal peptidase (Szabo et al., 2007), a homologue
of bacterial PilD, responsible for the processing of bacte-
rial type IV prepilin proteins (Strom et al., 1993). Indeed,
H. volcanii cells deleted of pibD (HVO_2993), encoding
the prepilin peptidase, fail to properly process proteins
bearing this class of signal peptide (Tripepi et al., 2010).
Lipid modification
Lipid modification of a protein involves the permanent
or temporary covalent attachment of lipid-based groups
at various positions within the polypeptide chain and
serves a variety of roles, with the most obvious being to
enhance the membrane affinity of the modified protein.
However, numerous other roles have been assigned to
such PTM, including modulation of protein–proteininteractions, signal transduction, embryogenesis, pattern
formation, protein trafficking through the secretory path-
way, evasion of the immune response by infectious para-
sites, and enzyme activation (for review, see Sinensky,
2000; Mann & Beachy, 2004; Pechlivanis & Kuhlmann,
2006; Nadolski & Linder, 2007; Charollais & Van Der
Goot, 2009; Greaves et al., 2009; Aicart-Ramos et al.,
2011). In efforts aimed at understanding the modes, path-
ways, and functions of lipid modification in archaea,
studies on H. volcanii have proven to be central.
In addressing the biogenesis of the H. volcanii S-layer
glycoprotein, it was shown that the protein is first
Fig. 7. Schematic depiction of haloarchaeal signal peptides and their processing. Type I and type II signal peptides that target proteins to the Sec
or Tat translocation pathways, as well as type III signal peptides, have been reported in haloarchaea. In each signal peptide, the light blue N-
terminal region contains positive charges (+) in the case of Sec pathway substrates or the twin arginine residues (RR) characteristic of Tat
pathway substrates. Type I signal peptidases cleave the signal peptide after a C-terminal region (dark blue) often ending in alanine(s). Type II
signal peptidases cleave the signal peptide upstream of the lipobox cysteine found in the LAGC consensus sequence. The exposed cysteine that
becomes the N-terminal residue of the mature protein may become lipid-modified. Type III signal peptidases act on a C-terminal domain
upstream of a hydrophobic stretch (sky blue). The cleavage sites processed by the various signal peptidases are indicated by black triangles. For
further details, see Pohlschroder et al. (2005).
FEMS Microbiol Rev 37 (2013) 583–606 ª 2012 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved
Protein modification in the haloarchaea 595
synthesized as an immature precursor, possessing a lower
apparent molecular weight and a less hydrophobic char-
acter than the final version of the protein (Eichler,
2001). As the protein can be labeled with [3H] mevalon-
ic acid, an isoprene precursor, the post-translational
magnesium-dependent conversion to the mature form
apparently involves isoprenylation (Konrad & Eichler,
2002). Moreover, it was shown that such lipid modifica-
tion of the H. volcanii S-layer glycoprotein only occurs
once the protein has traversed the plasma membrane
(Eichler, 2001). Similarly, the H. salinarum S-layer glyco-
protein also undergoes such maturation and was shown
to be modified by a covalently linked diphytanylglyceryl
phosphate entity near an O-glycosylated Thr-rich stretch
found in the C-terminal region of the protein, upstream
of the predicted single transmembrane domain (Kikuchi
et al., 1999; Konrad & Eichler, 2002). At the same time,
several H. salinarum proteins are modified by a diphyta-
nylglycerol methyl moiety, linked to Cys residues of the
protein through a thioetheric bond (Sagami et al., 1994,
1995).
As the H. volcanii S-layer glycoprotein includes a pre-
dicted membrane-spanning domain (as does its H. salina-
rum counterpart) (Lechner & Sumper, 1987; Sumper
et al., 1990), it is unclear why an additional membrane
anchor in the form of a lipid would be required. The
recent identification of genes encoding archaeosortases
may explain this apparent paradox (Haft et al., 2012).
Archaeosortases are predicted by bioinformatics to corre-
spond to the archaeal homologues of bacterial exosortases,
enzymes involved in C-terminal anchoring of proteins to
membrane-embedded lipids. The archaeosortase, ArtA, is
predicted to cleave proteins bearing a C-terminal mem-
brane-spanning domain between this region and the Pro-
Gly-Phe motif found immediately upstream. The
processed protein is then delivered to the waiting lipid on
the external surface of the cell. Accordingly, the H. volca-
nii S-layer glycoprotein (and its H. salinarum counterpart;
Lechner & Sumper, 1987) present a Pro-Gly-Phe motif
found immediately upstream of the predicted transmem-
brane domain (Sumper et al., 1990). Indeed, the coordi-
nated transfer of the H. volcanii S-layer glycoprotein to a
lipid moiety following cleavage from the C-terminal mem-
brane-spanning domain could explain several apparent
paradoxes, such as the ability of EDTA to solubilize the
S-layer glycoprotein, an apparent integral membrane pro-
tein, without destroying membrane integrity (Charlebois
et al., 1987). In this scenario, EDTA treatment would
somehow interfere with the stabilization of newly lipid-
modified S-layer glycoprotein in the membrane or in the
S-layer, leading to the release of the protein from the cell
while leaving the plasma membrane intact. Most recently,
it has been reported that two H. volcanii S-layer glycopro-
tein populations exist, namely a lipid-modified, EDTA-
solublized pool and a second, detergent-solublized pool
likely retaining the transmembrane domain of the protein
(Kandiba et al., 2013).
In addition to such C-terminal post-translational lipid
modification, N-terminal lipid modification also occurs,
namely in processing of lipoproteins. Based on the pres-
ence the so-called lipobox sequence motif (composed of
the consensus Leu-Ala-Gly-Cys sequence) that includes
the cysteine residue that undergoes lipid modification
(Hayashi & Wu, 1990) near the start of predicted amino
acid sequence, halocyanin, a small blue copper protein of
the haloalkaliphile N. pharaonis is thought to undergo
such amino-terminal lipid modification (Mattar et al.,
1994). Although direct proof for such modification was
not provided, analysis of halocyanin by mass spectroscopy
was consistent with modification of the N-terminal Cys
residue by two C20 phytanyl (glycerol)diether lipids linked
via a thioether bond, as well as by acetylation. Indeed,
analysis of haloarchaeal genomes predicts numerous lipo-
box-containing proteins processed by the Tat protein
translocation system. In H. volcanii, 72 lipobox-contain-
ing proteins are predicted (Gim�enez et al., 2007). In the
specific cases of the H. volcanii iron-binding protein
(Ibp), DsbA-like thioredoxin domain protein (DsbA), and
maltose-binding protein (Mbp), replacement of the lipo-
box cysteine by serine in the precursor forms of these
predicted lipoproteins led to defective localization.
Mutant Ibp and DsbA were found in the growth medium
instead of being largely membrane associated, as are the
wild-type proteins; mutant Mpb is apparently unstable
and degraded (Gim�enez et al., 2007). On the other hand,
the predicted lipoproteins, HVO_B0139 and HVO_1242,
remained cell-associated in the lipobox cysteine-alanine
mutated forms (Storf et al., 2010).
The lipid-based modifications considered above essen-
tially serve to anchor the modified protein to the mem-
brane. The retinal proteins of haloarchaea (Oesterhelt &
Stoeckenius, 1971) provide another use for covalently
linked lipids. Bacteriorhodopsin, halorhodopsin, and the
two sensory rhodopsins [and related rhodopsins subse-
quently reported in Bacteria (Beja et al., 2000) and
eukaryotes (Bieszke et al., 1999)], comprise a family of
small, seven trans-membrane domain-containing proteins
involved in coupling light energy to the vectorial trans-
port of ions across a membrane. To achieve this feat,
these retinal proteins contain covalently linked retinal, an
isoprene-derived light-sensitive co-factor. Accordingly,
bacteriorhodopsin, the prototype rhodopsin, has served as
a useful reporter of the assembly of lipid modification of
proteins. For more information on this and related top-
ics, the reader is directed to reviews by Lanyi (2004) and
Grote & O’Malley (2011).
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Rev 37 (2013) 583–606Published by John Wiley & Sons Ltd. All rights reserved
596 J. Eichler & J. Maupin-Furlow
Sampylation
SAMPs are small archaeal ubiquitin-like modifier proteins
that form isopeptide bonds to lysine residues of archaeal
proteins (Fig. 8). While this system was first demon-
strated in H. volcanii (Humbard et al., 2010a), it is
predicted to exist in all Archaea and appears related to
other recently discovered protein conjugation systems,
including the TtuBC system of protein conjugation in the
hyperthermophilic bacterium, Thermus thermophilus
(Humbard et al., 2010a; Shigi, 2012), and the Urm1 sys-
tem of protein conjugation and sulfur transfer in eukary-
otes (Wang et al., 2011). Sampylation is thought to be
reversible, based on conservation of JAMM (JAB1/MPN/
Mov34 metalloenzyme) domain homologs in all Archaea
(Humbard et al., 2010a; Maupin-Furlow, 2012). In
eukaryotes, JAMM domain proteins include the Rpn11/
Poh1 subunit of 26S proteasomes, required for removal
of ubiquitin chains from proteins (Verma et al., 2002;
Yao & Cohen, 2002). Likewise, Csn5/Jab1 is a JAMM
domain protein subunit of the COP9 signalosome com-
plex that cleaves the ubiquitin-like Nedd8 from proteins
(Cope et al., 2002). While archaeal JAMM domain pro-
tein function has yet to be demonstrated, the solved crys-
tal structure of an A. fulgidus JAMM domain protein
(termed AfJAMM) was used to model the structures of
Rpn11/Poh1 and Csn5/Jab1 (Tran et al., 2003; Ambroggio
et al., 2004).
Much like the ubiquitin and ubiquitin-like protein
modifiers of eukaryotes, SAMPs have a b-grasp fold
structure and C-terminal diglycine motif that is impor-
tant to their function in protein conjugation (Humbard
et al., 2010a; Ranjan et al., 2010; Jeong et al., 2011). The
a-carboxyl of the C-terminal glycine of SAMPs can form
an isopeptide bond to the e-amino group of lysine resi-
dues of target proteins by a mechanism that requires
UbaA, an ubiquitin-activating E1 enzyme homolog of
Archaea (Humbard et al., 2010a; Miranda et al., 2011).
The physiological reason for the formation of these
ubiquitin-like protein conjugates in Archaea (and hyper-
thermophilic bacteria) remains to be determined. How-
ever, in analogy to ubiquitin, sampylation is thought to
alter the structure, enzymatic activity, and types of part-
ners that would associate with the modified protein,
including proteasomes, transcription factors, and others
(Maupin-Furlow, 2012). Interestingly, SAMPs not only
form isopeptide bonds with protein targets but also are
required for sulfur transfer to biomolecules, including
molybdenum cofactor (MoCo) and tRNA (similar to
Urm1 of eukaryotes and TtuB of the bacterium T. ther-
mophilus) (Miranda et al., 2011; Shigi, 2012). Sampyla-
tion differs from the system of protein conjugation
recently predicted for the archaeon Candidatus Caldiar-
chaeum subterraneum (based on metagenomics) (Nunoura
et al., 2011) and some bacteria (based on analogy) (Burr-
oughs et al., 2011). This latter system is rare in Archaea
(restricted to Candidatus C. subterraneum) and appears
to incorporate not only ubiquitin-activating E1 homologs
but also ubiquitin-conjugating E2 and ubiquitin ligase E3
homologs (Nunoura et al., 2011).
-C-OH
ATP PPi
C-terminalα-carboxyl group
(Gly residue)
Acyl-adenylate
-C-AMP
Isopeptide-linkedprotein conjugate
Thioester
SAMP JAMM
AMP
UbaA
SAMPSAMP
SAMP
Sulfur transferpathways
=O =
O
UbaA
SH
Cys188sulfhydryl
groupSC=O N-H
C=O
UbaA
Substrateprotein
Substrateprotein
+NH3Lysε-aminogroup
Protease
-C-S-XSAMP
=
O
Sulfur containing biomolecules(e.g. MoCo, thiolated tRNA)
Specificityfactors fortargeting?
Fig. 8. Sampylation in Haloferax volcanii. The H. volcanii ubiquitin-like SAMP1/2 and UbaA (a ubiquitin-activating E1 enzyme homolog) function
in protein conjugation (sampylation) and sulfur transfer (biosynthesis of MoCo and thiolated tRNA). In these pathways, UbaA is thought to
adenylate the C-terminal glycine of the SAMP1/2. In protein conjugation, a thioester intermediate is thought to form between a conserved active
site cysteine of UbaA (C188) and the C-terminal carboxyl group of the SAMP. SAMP1/2 are then transferred to lysine residues on protein targets
to form an isopeptide bond. Archaeal proteins of the Jab1/Mov34/Mpr1 Pad1 N-terminal+ (MPN+) (JAMM) domain superfamily are proposed to
cleave isopeptide bonds and, thus, render the pathway reversible.
FEMS Microbiol Rev 37 (2013) 583–606 ª 2012 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved
Protein modification in the haloarchaea 597
Protein degradation
Proteases are important in maintaining cellular homeosta-
sis, regulating cellular signaling, and degrading exogenous
proteins for cellular metabolism. In these processes, pro-
teases are needed to catalyze the general turnover of
proteins, remove aberrant proteins (e.g. damaged or
viral-encoded proteins), and mediate the precisely timed
cleavage or turnover of regulatory proteins important to
cell functions ranging from transcription and cell division
to metabolism (Gottesman, 2003). Similarly to other
archaea, H. volcanii is predicted to encode a wide variety
of endo- and exo-proteases, including energy-dependent
and intramembrane-cleaving proteases, typically linked to
the early events of protein degradation and regulation
(Maupin-Furlow et al., 2005; De Castro et al., 2006). Of
the energy-dependent proteases, proteasomes and mem-
brane-associated Lon B-type proteases appear relatively
universal in the Archaea (including H. volcanii) (Cha
et al., 2010; Maupin-Furlow, 2012).
Among the various regulatory proteases (i.e. excluding
signal peptidases) predicted for H. volcanii, only the pro-
teasomes have been studied at the biochemical level (Mau-
pin-Furlow, 2012). 20S proteasomes are purified from
H. volcanii in at least three distinct subtypes, composed of
four stacked heptameric rings of different subunit compo-
sition (i.e. a17b7b7a17, a17b7b7a27, and a27b7b7a27,where subscript represents subunit stoichiometry) (Wilson
et al., 1999; Kaczowka & Maupin-Furlow, 2003; Karadzic
et al., 2012). The 20S proteasomes are catalytically active
in cleaving peptides and small denatured proteins (i.e. oxi-
dized bovine insulin B-chain protein) (Wilson et al., 1999;
Kaczowka & Maupin-Furlow, 2003; Karadzic et al., 2012).
Haloferax volcanii PANs are purified as PAN-A and
PAN-B subclasses but were not found to be active in stim-
ulating the energy-dependent degradation of proteins by
20S proteasomes (based on in vitro assay) (Reuter et al.,
2004; Humbard et al., 2010b). The levels of the a2 subunit
of 20S proteasomes and PAN-B are correlated with growth
phase (i.e. low levels detected in lag to log phase and high
levels detected in stationary phase) suggesting that these
two proteins are associated in a biologically related path-
way (Reuter et al., 2004).
Proteasomes appear important in cell division, protein
quality control, and other functions in H. volcanii. Like
eukaryotes, 20S proteasomes of H. volcanii are essential,
based on the conditional lethal phenotypes associated
with deletion of the b gene or double deletion of the a1and a2 genes (Zhou et al., 2008). The PANs are not
essential (panA panB double mutants are viable) (Zhou
et al., 2008), while Cdc48d (one of four Cdc48 homologs
in H. volcanii) appears essential, based on the inability to
delete this gene from the chromosome (Allers et al.,
2010). PAN, Cdc48/p97/VAT, and other members of the
AAA ATPase superfamily are thought to form networks
of ATPases that associate with and regulate the function
of 20S proteasomes in archaeal cells (Barthelme & Sauer,
2012; Forouzan et al., 2012; Maupin-Furlow, 2012). Thus,
Cdc48d and 20S proteasomes may regulate functions of
cell division. Consistent with a role in protein quality
control, proteasomes (PAN-A and a1) are needed for
H. volcanii to tolerate stressful conditions, including
exposure to L-canavanine (an amino acid analogue that
causes protein unfolding) and low salt (Zhou et al.,
2008). In addition, a1 is needed to overcome thermal
stress (Zhou et al., 2008). Whether sampylation is linked
to proteasome-mediated proteolysis, in analogy to the
ubiquitin-proteasome system of eukaryotic cells, remains
to be determined. Levels of SAMP1-protein conjugates
are, however, higher in proteasome mutants, as compared
to wild-type cells, providing indirect evidence for a con-
nection between these two systems (Humbard et al.,
2010a). Still, ubaA (encoding the single ubiquitin-activat-
ing E1 homolog) can be deleted without loss of cell
viability, suggesting that sampylation is not required for
cell division in H. volcanii (Miranda et al., 2011).
Conclusions
Across evolution, proteins can experience any of a long list
of PTMs that lead to numerous and varied effects on pro-
tein structure, function, localization, oligomerization sta-
tus, and stability. As is the case for much of archaeal
biology, the various PTMs performed by Archaea both
present traits unique to this life form and others shared by
eukaryotes and/or Bacteria. As presented in Table 2, post-
Table 2. List of some PTMs reported in Haloferax volcanii
PTM Experimentally-verified target(s)
N-glycosylation S-layer glycoprotein (HVO_2072),
FlgA1 (HVO_1210)
O-glycosylation S-layer glycoprotein (HVO_2072)
X-glycosylation* 150, 105, 98, 58, 56, 54 and
52 kDa proteins
Lipid modification S-layer glycoprotein (HVO_2072)
Signal peptide cleavage S-layer glycoprotein (HVO_2072),
FlgA1 (HVO_1210), FlgA2 (HVO_1211)
Sampylation SAMP2 (HVO_0202), UbaA, homologs
of ribose-1,5-bisphosphate isomerase
(HVO_0966), DNA gyrase B subunit
(HVO_1572), cysteine hydrolyase
(HVO_2328), NADH-quinone
oxidoreductase chain c/d (HVO_0980),
OsmC (HVO_1289), TBPe (HVO_1727), and
tandem rhodanese domain protein
(HVO_0025)
*X-glycosylation, form of glycosylation undetermined.
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Rev 37 (2013) 583–606Published by John Wiley & Sons Ltd. All rights reserved
598 J. Eichler & J. Maupin-Furlow
translationally modified proteins abound in H. volcanii,
justifying the use of this species for the study of the
responsible processes in archaea. With an ever-growing list
of tools available for the genetic, proteomic, biochemical,
and physiological examination of H. volcanii, it is likely
that future studies on this organism, and indeed, on other
haloarchaea, will continue to provide novel insight into
PTMs in general, and in archaea, in particular.
Acknowledgements
J.E. is supported by grants from the Israel Science Foun-
dation (8/11) and the US Army Research Office
(W911NF-11-1-520). J.M.-F. is supported by grants from
NIH (R01 GM057498) and DOE (DE-FG02-05ER15650).
References
Abe Y, Matsumoto S, Wei S et al. (2001) Cloning and
characterization of a p53-related protein kinase expressed in
interleukin-2-activated cytotoxic T-cells, epithelial tumor
cell lines, and the testes. J Biol Chem 276: 44003–44011.Abu-Qarn M & Eichler J (2006) Protein N-glycosylation in
Archaea: defining Haloferax volcanii genes involved in S-layer
glycoprotein glycosylation. Mol Microbiol 61: 511–525.Abu-Qarn M, Yurist-Doutsch S, Giordano A, Trauner A,
Morris HR, Hitchen P, Medalia O, Dell A & Eichler J
(2007) Haloferax volcanii AglB and AglD are involved in
N-glycosylation of the S-layer glycoprotein and proper
assembly of the surface layer. J Mol Biol 14: 1224–1236.Abu-Qarn M, Giordano A, Battaglia F, Trauner A, Morris HR,
Hitchen P, Dell A & Eichler J (2008a) Identification of
AglE, a second glycosyltransferase involved in
N-glycosylation of the Haloferax volcanii S-layer
glycoprotein. J Bacteriol 190: 3140–3146.Abu-Qarn M, Eichler J & Sharon N (2008b) Not just for
Eukarya anymore: N-glycosylation in Bacteria and Archaea.
Curr Opin Struct Biol 18: 544–550.Aicart-Ramos C, Valero RA & Rodriguez-Crespo I (2011)
Protein palmitoylation and subcellular trafficking. Biochim
Biophys Acta 1808: 2981–2994.Aivaliotis M, Gevaert K, Falb M et al. (2007) Large-scale
identification of N-terminal peptides in the halophilic
archaea Halobacterium salinarum and Natronomonas
pharaonis. J Proteome Res 6: 2195–2204.Aivaliotis M, Macek B, Gnad F, Reichelt P, Mann M &
Oesterhelt D (2009) Ser/Thr/Tyr protein phosphorylation in
the archaeon Halobacterium salinarum–a representative of
the third domain of life. PLoS ONE 4: e4777.
Aka JA, Kim GW & Yang XJ (2011) K-acetylation and its
enzymes: overview and new developments. Handb Exp
Pharmacol 206: 1–12.Alam M, Lebert M, Oesterhelt D & Hazelbauer GL (1989)
Methyl-accepting taxis proteins in Halobacterium halobium.
EMBO J 8: 631–639.
Albers SV, Szabo Z & Driessen AJM (2003) Archaeal homolog
of bacterial type IV prepilin signal peptidases with broad
substrate specificity. J Bacteriol 185: 3918–3925.Allers T, Ngo HP, Mevarech M & Lloyd RG (2004)
Development of additional selectable markers for the
halophilic archaeon Haloferax volcanii based on the leuB
and trpA genes. Appl Environ Microbiol 70: 943–953.Allers T, Barak S, Liddell S, Wardell K & Mevarech M (2010)
Improved strains and plasmid vectors for conditional
overexpression of His-tagged proteins in Haloferax volcanii.
Appl Environ Microbiol 76: 1759–1769.Altman-Price N & Mevarech M (2009) Genetic evidence for
the importance of protein acetylation and protein
deacetylation in the halophilic archaeon Haloferax volcanii.
J Bacteriol 191: 1610–1617.Ambroggio XI, Rees DC & Deshaies RJ (2004) JAMM: a
metalloprotease-like zinc site in the proteasome and
signalosome. PLoS Biol 2: E2.
Arfin SM, Kendall RL, Hall L, Weaver LH, Stewart AE,
Matthews BW & Bradshaw RA (1995) Eukaryotic methionyl
aminopeptidases: two classes of cobalt-dependent enzymes.
P Natl Acad Sci USA 92: 7714–7718.Barabote RD & Saier MH Jr (2005) Comparative genomic
analyses of the bacterial phosphotransferase system.
Microbiol Mol Biol Rev 69: 608–634.Bardy SL, Ng SY, Carnegie DS & Jarrell KF (2005) Site-
directed mutagenesis analysis of amino acids critical for
activity of the type I signal peptidase of the archaeon
Methanococcus voltae. J Bacteriol 187: 1188–1191.Barthelme D & Sauer RT (2012) Identification of the
Cdc48*20S proteasome as an ancient AAA+ proteolytic
machine. Science 337: 843–846.Bartig D, Lemkemeier K, Frank J, Lottspeich F & Klink F
(1992) The archaebacterial hypusine-containing protein.
Structural features suggest common ancestry with
eukaryotic translation initiation factor 5A. Eur J Biochem
204: 751–758.Bazan JF, Weaver LH, Roderick SL, Huber R & Matthews BW
(1994) Sequence and structure comparison suggest that
methionine aminopeptidase, prolidase, aminopeptidase P,
and creatinase share a common fold. P Natl Acad Sci USA
91: 2473–2477.Beja O et al. (2000) Bacterial rhodopsin: evidence for a new
type of phototrophy in the sea. Science 289: 1902–1906.Bell SD, Botting CH, Wardleworth BN, Jackson SP & White
MF (2002) The interaction of Alba, a conserved archaeal
chromatin protein, with Sir2 and its regulation by
acetylation. Science 296: 148–151.Bieszke JA, Spudich EN, Scott KL, Borkovich KA & Spudich
JL (1999) A eukaryotic protein, NOP-1, binds retinal to
form an archaeal rhodopsin-like photochemically reactive
pigment. Biochemistry 38: 14138–14145.Brooun A, Zhang W & Alam M (1997) Primary structure and
functional analysis of the soluble transducer protein HtrXI
in the archaeon Halobacterium salinarium. J Bacteriol 179:
2963–2968.
FEMS Microbiol Rev 37 (2013) 583–606 ª 2012 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved
Protein modification in the haloarchaea 599
Burda P & Aebi M (1999) The dolichol pathway of N-linked
glycosylation. Biochim Biophys Acta 1426: 239–257.Burroughs AM, Iyer LM & Aravind L (2011) Functional
diversification of the RING finger and other binuclear treble
clef domains in prokaryotes and the early evolution of the
ubiquitin system. Mol BioSyst 7: 2261–2277.Calo D & Eichler J (2011) Crossing the membrane in Archaea,
the third domain of life. Biochim Biophys Acta 1808: 885–901.Calo D, Eilam Y, Lichtenstein RG & Eichler J (2010b) Towards
glyco-engineering in Archaea: replacing Haloferax volcanii
AglD with homologous glycosyltransferases from other
halophilic archaea. Appl Environ Microbiol 76: 5684–5692.Calo D, Guan Z & Eichler J (2011) Glyco-engineering in
Archaea: differential N-glycosylation of the S-layer
glycoprotein in a transformed Haloferax volcanii strain.
Microb Biotechnol 4: 461–470.Cha SS, An YJ, Lee CR et al. (2010) Crystal structure of Lon
protease: molecular architecture of gated entry to a
sequestered degradation chamber. EMBO J 29: 3520–3530.Chaban B, Voisin S, Kelly J, Logan SM & Jarrell KF (2006)
Identification of genes involved in the biosynthesis and
attachment of Methanococcus voltae N-linked glycans:
insight into N-linked glycosylation pathways in Archaea.
Mol Microbiol 61: 259–268.Charlebois RL, Lam WL, Cline SW & Doolittle WF (1987)
Characterization of pHV2 from Halobacterium volcanii and
its use in demonstrating transformation of an
archaebacterium. P Natl Acad Sci USA 84: 8530–8534.Charollais J & Van Der Goot FG (2009) Palmitoylation of
membrane proteins. Mol Membr Biol 26: 55–66.Chen G, Liu H, Wang X & Li Z (2010) In vitro methylation by
methanol: proteomic screening and prevalence investigation.
Anal Chim Acta 661: 67–75.Clarke S (1993) Protein methylation. Curr Opin Cell Biol 5:
977–983.Cohen-Rosenzweig C, Yurist-Doutsch S & Eichler J (2012)
AglS, a novel component of the Haloferax volcanii
N-glycosylation pathway, is a dolichol phosphate-mannose
mannosyltransferase. J Bacteriol 194: 6909–6916.Cope GA, Suh GS, Aravind L, Schwarz SE, Zipursky SL,
Koonin EV & Deshaies RJ (2002) Role of predicted
metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8
from Cul1. Science 298: 608–611.DasSarma S & Fleishmann EM (1995) Archaea: A Laboratory
Manual – Halophiles. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY.
De Castro RE, Maupin-Furlow JA, Gimenez MI, Herrera Seitz
MK & Sanchez JJ (2006) Haloarchaeal proteases and
proteolytic systems. FEMS Microbiol Rev 30: 17–35.Dilks K, Rose RW, Hartmann E & Pohlschroder M (2003)
Prokaryotic utilization of the twin-arginine
translocation pathway: a genomic survey. J Bacteriol
185: 1478–1483.Dyall-Smith M (2009) The Halohandbook: Protocols for
Halobacterial Genetics. http://www.haloarchaea.com/
resources/handbook/Halohandbook_2009_v7.1.pdf.
Eichler J (2000) Novel glycoproteins of the halophilic archaeon
Haloferax volcanii. Arch Microbiol 173: 445–448.Eichler J (2001) Post-translational modification of the S-layer
glycoprotein occurs following translocation across the
plasma membrane of the haloarchaeon Haloferax volcanii.
Eur J Biochem 268: 4366–4373.Eichler J & Adams MW (2005) Posttranslational protein
modification in Archaea. Microbiol Mol Biol Rev 69:
393–425.Enoki M, Shinzato N, Sato H, Nakamura K & Kamagata Y
(2011) Comparative proteomic analysis of
Methanothermobacter themautotrophicus DH in pure culture
and in co-culture with a butyrate-oxidizing bacterium. PLoS
ONE 6: e24309.
Evans EA, Gilmore R & Blobel G (1986) Purification of
microsomal signal peptidase as a complex. P Natl Acad Sci
USA 83: 581–585.Ewens CA, Kloppsteck P, Forster A, Zhang X & Freemont PS
(2010) Structural and functional implications of
phosphorylation and acetylation in the regulation of the
AAA+ protein p97. Biochem Cell Biol 88: 41–48.Facchin S, Lopreiato R, Ruzzene M, Marin O, Sartori G, Gotz C,
Montenarh M, Carignani G & Pinna LA (2003) Functional
homology between yeast piD261/Bud32 and human PRPK:
both phosphorylate p53 and PRPK partially complements
piD261/Bud32 deficiency. FEBS Lett 549: 63–66.Falb M, Aivaliotis M, Garcia-Rizo C, Bisle B, Tebbe A, Klein
C, Konstantinidis K, Siedler F, Pfeiffer F & Oesterhelt D
(2006) Archaeal N-terminal protein maturation commonly
involves N-terminal acetylation: a large-scale proteomics
survey. J Mol Biol 362: 915–924.Fass D (2011) Disulfide bonding in protein biophysics. Annu
Rev Biophys, 41: 63–79.Fine A, Irihimovitch V, Dahan I, Konrad Z & Eichler J (2006)
Cloning, expression and purification of functional Sec11a
and Sec11b, type I signal peptidases of the archaeon
Haloferax volcanii. J Bacteriol 188: 1911–1919.Fink-Lavi E & Eichler J (2008) Identification of residues
essential for the catalytic activity of Sec11b, one of the two
type I signal peptidases of Haloferax volcanii. FEMS
Microbiol Lett 278: 257–260.Fischer C, Geourjon C, Bourson C & Deutscher J (1996)
Cloning and characterization of the Bacillus subtilis prkA gene
encoding a novel serine protein kinase. Gene 168: 55–60.Forbes AJ, Patrie SM, Taylor GK, Kim YB, Jiang L & Kelleher
NL (2004) Targeted analysis and discovery of
posttranslational modifications in proteins from
methanogenic archaea by top-down MS. P Natl Acad Sci
USA 101: 2678–2683.Forouzan D, Ammelburg M, Hobel CF, Stroh LJ, Sessler N,
Martin J & Lupas AN (2012) The archaeal proteasome is
regulated by a network of AAA ATPases. J Biol Chem 287:
39254–39262.Gil MA, Sherwood KE & Maupin-Furlow JA (2007)
Transcriptional linkage of Haloferax volcanii proteasomal
genes with non-proteasomal gene neighbours including
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Rev 37 (2013) 583–606Published by John Wiley & Sons Ltd. All rights reserved
600 J. Eichler & J. Maupin-Furlow
RNase P, MOSC domain and SAM-methyltransferase
homologues. Microbiology 153: 3009–3022.Gim�enez MI, Dilks K & Pohlschr€oder M (2007) Haloferax
volcanii twin-arginine translocation substates include
secreted soluble, C-terminally anchored and lipoproteins.
Mol Microbiol 66: 1597–1606.Gogarten JP, Senejani AG, Zhaxybayeva O, Olendzenski L &
Hilario E (2002) Inteins: structure, function, and evolution.
Annu Rev Microbiol 56: 263–287.Gottesman S (2003) Proteolysis in bacterial regulatory circuits.
Annu Rev Cell Dev Biol 19: 565–587.Greaves J, Prescott GR, Gorleku OA & Chamberlain LH
(2009) The fat controller: roles of palmitoylation in
intracellular protein trafficking and targeting to membrane
microdomains. Mol Membr Biol 26: 67–79.Grote M & O’Malley MA (2011) Enlightening the life sciences:
the history of halobacterial and microbial rhodopsin
research. FEMS Microbiol Rev 35: 1082–1099.Guan Z, Naparstek S, Kaminski L, Konrad Z & Eichler J
(2010) Distinct glycan-charged phosphodolichol carriers are
required for the assembly of the pentasaccharide N-linked
to the Haloferax volcanii S-layer glycoprotein. Mol Microbiol
78: 1294–1303.Guan Z, Naparstek S, Calo D & Eichler J (2012) Protein
glycosylation as an adaptive response in Archaea: growth at
different salt concentrations leads to alterations in Haloferax
volcanii S-layer glycoprotein N-glycosylation. Environ
Microbiol 14: 743–753.Guillon JM, Mechulam Y, Schmitter JM, Blanquet S & Fayat G
(1992) Disruption of the gene for Met-tRNA(fMet)
formyltransferase severely impairs growth of Escherichia coli.
J Bacteriol 174: 4294–4301.Haft DH, Payne SH & Selengut JD (2012) Archaeosortases and
exosortases are widely distributed systems linking membrane
transit with posttranslational modification. J Bacteriol 194:
36–48.Haile JD & Kennelly PJ (2011) The activity of an ancient
atypical protein kinase is stimulated by ADP-ribose in vitro.
Arch Biochem Biophys 511: 56–63.Harris RA, Popov KM, Zhao Y, Kedishvili NY, Shimomura Y &
Crabb DW (1995) A new family of protein kinases–themitochondrial protein kinases. Adv Enzyme Regul 35: 147–162.
Hartley MD & Imperiali B (2012) At the membrane frontier: a
prospectus on the remarkable evolutionary conservation of
polyprenols and polyprenyl-phosphates. Arch Biochem
Biophys 517: 83–97.Hartman AL, Norais C, Badger JH et al. (2010) The complete
genome sequence of Haloferax volcanii DS2, a model
archaeon. PLoS ONE 5: e9605.
Hase T, Wakabayashi S, Matsubara H, Kerscher L,
Oesterhelt D, Rao KK & Hall DO (1978) Complete
amino acid sequence of Halobacterium halobium
ferredoxin containing an Ne-acetyllysine residue.
J Biochem 83: 1657–1670.Hase T, Wakabayashi S, Matsubara H, Mevarech M & Werber
MM (1980) Amino acid sequence of 2Fe-2S ferredoxin from
an extreme halophile, Halobacterium of the Dead Sea.
Biochim Biophys Acta 623: 139–145.Hatakeyama T & Hatakeyama T (1990) Amino acid sequences
of the ribosomal proteins HL30 and HmaL5 from the
archaebacterium Halobacterium marismortui. Biochim
Biophys Acta 1039: 343–347.Hayashi S & Wu HC (1990) Lipoproteins in bacteria.
J Bioenerg Biomembr 22: 451–471.Hecker A, Lopreiato R, Graille M, Collinet B, Forterre P, Libri
D & van Tilbeurgh H (2008) Structure of the archaeal Kae1/
Bud32 fusion protein MJ1130: a model for the eukaryotic
EKC/KEOPS subcomplex. EMBO J 27: 2340–2351.Hegde RS & Bernstein HD (2006) The surprising complexity
of signal sequences. Trends Biochem Sci 31: 563–571.Helbig AO, Gauci S, Raijmakers R, van Breukelen B, Slijper M,
Mohammed S & Heck AJ (2010) Profiling of N-acetylated
protein termini provides in-depth insights into the
N-terminal nature of the proteome. Mol Cell Proteomics 9:
928–939.Helenius A & Aebi M (2004) Roles of N-linked glycans in the
endoplasmic reticulum. Annu Rev Biochem 73: 1019–1049.Hu LI, Lima BP & Wolfe AJ (2010) Bacterial protein acetylation:
the dawning of a new age. Mol Microbiol 77: 15–21.Humbard MA, Stevens SM Jr & Maupin-Furlow JA (2006)
Posttranslational modification of the 20S proteasomal
proteins of the archaeon Haloferax volcanii. J Bacteriol 188:
7521–7530.Humbard MA, Zhou G & Maupin-Furlow JA (2009) The
N-terminal penultimate residue of 20S proteasome a1influences its Na-acetylation and protein levels as well as
growth rate and stress responses of Haloferax volcanii.
J Bacteriol 191: 3794–3803.Humbard MA, Reuter CJ, Zuobi-Hasona K, Zhou G &
Maupin-Furlow JA (2010a) Phosphorylation and
methylation of proteasomal proteins of the haloarcheon
Haloferax volcanii. Archaea 2010: 481725.
Humbard MA, Miranda HV, Lim JM, Krause DJ, Pritz JR,
Zhou G, Chen S, Wells L & Maupin-Furlow JA (2010b)
Ubiquitin-like small archaeal modifier proteins (SAMPs) in
Haloferax volcanii. Nature 463: 54–60.Hwang CS, Shemorry A & Varshavsky A (2010) N-terminal
acetylation of cellular proteins creates specific degradation
signals. Science 327: 973–977.Igura M, Maita N, Kamishikiryo J, Yamada M, Obita T,
Maenaka K & Kohda D (2008) Structure-guided
identification of a new catalytic motif of
oligosaccharyltransferase. EMBO J 27: 234–243.Jarrell KF & Albers SV (2012) The archaellum: an old motility
structure with a new name. Trends Microbiol 20: 307–312.Jarrell KF, Jones GM, Kandiba L, Nair DB & Eichler J (2010)
S-layer glycoproteins and flagellins: reporters of archaeal
post-translational modifications. Archaea 2010: 612948.
Jarvis P & Robinson C (2004) Mechanisms of protein import
and routing in chloroplasts. Curr Biol 14: R1064–R1077.Jelinska C, Conroy MJ, Craven CJ, Hounslow AM, Bullough
PA, Waltho JP, Taylor GL & White MF (2005) Obligate
FEMS Microbiol Rev 37 (2013) 583–606 ª 2012 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved
Protein modification in the haloarchaea 601
heterodimerization of the archaeal Alba2 protein with Alba1
provides a mechanism for control of DNA packaging.
Structure 13: 963–971.Jeong YJ, Jeong BC & Song HK (2011) Crystal structure of
ubiquitin-like small archaeal modifier protein 1 (SAMP1)
from Haloferax volcanii. Biochem Biophys Res Commun 405:
112–117.Johnson LN & Barford D (1993) The effects of
phosphorylation on the structure and function of proteins.
Annu Rev Biophys Biomol Struct 22: 199–232.Jones JD & O’Connor CD (2011) Protein acetylation in
prokaryotes. Proteomics 11: 3012–3022.Jones GM, Wu J, Ding Y, Uchida K, Aizawa S, Robotham A,
Logan SM, Kelly J & Jarrell KF (2012) Identification of
genes involved in the acetamidino group modification of
the flagellin N-linked glycan of Methanococcus maripaludis.
J Bacteriol 194: 2693–2702.Kaczowka SJ & Maupin-Furlow JA (2003) Subunit topology of
two 20S proteasomes from Haloferax volcanii. J Bacteriol
185: 165–174.Kaminski L, Abu-Qarn M, Guan Z, Naparstek S, Ventura VV,
Raetz CRH, Hitchen PG, Dell A & Eichler J (2010) AglJ
adds the first sugar of the N-linked pentasaccharide
decorating the Haloferax volcanii S-layer glycoprotein.
J Bacteriol 192: 5572–5579.Kaminski L, Guan Z, Abu-Qarn M, Konrad Z & Eichler J
(2012) AglR is required for addition of the final mannose
residue of the N-linked glycan decorating the Haloferax
volcanii S-layer glycoprotein. Biochim Biophys Acta 1820:
1664–1670.Kandiba L, Aitio O, Helin J, Guan Z, Permi P, Bamford D,
Eichler J & Roine E (2012) Diversity in prokaryotic
glycosylation: an archaeal-derived N-linked glycan contains
legionaminic acid. Mol Microbiol 84: 578–593.Kandiba L, Guan Z & Eichler J (2013) Lipid modification
gives rise to two distinct Haloferax volcanii S-layer
glycoprotein populations. Biochim Biophys Acta,
in press.
Karadzic I, Maupin-Furlow J, Humbard M, Singh P &
Goodlett D (2012) Chemical cross-linking, mass
spectrometry and in silico modeling of proteasomal 20S core
particles of the haloarchaeon Haloferax volcanii. Proteomics
12: 1806–1814.Kennelly PJ (2003) Archaeal protein kinases and protein
phosphatases: insights from genomics and biochemistry.
Biochem J 370: 373–389.Kennelly PJ & Potts M (1999) Life among the primitives:
protein O-phosphatases in prokaryotes. Front Biosci 4:
D372–D385.Kikuchi A, Sagami H & Ogura K (1999) Evidence for covalent
attachment of diphytanylglyceryl phosphate to the cell-
surface glycoprotein of Halobacterium halobium. J Biol Chem
274: 18011–18016.Kim D & Forst S (2001) Genomic analysis of the histidine
kinase family in bacteria and archaea. Microbiology 147:
1197–1212.
Kimura M, Arndt E, Hatakeyama T, Hatakeyama T & Kimura
J (1989) Ribosomal proteins in Halobacteria. Can
J Microbiol 35: 195–199.Kirkland PA, Gil MA, Karadzic IM & Maupin-Furlow JA
(2008a) Genetic and proteomic analyses of a proteasome-
activating nucleotidase A mutant of the haloarchaeon
Haloferax volcanii. J Bacteriol 190: 193–205.Kirkland PA, Humbard MA, Daniels CJ & Maupin-Furlow JA
(2008b) Shotgun proteomics of the haloarchaeon Haloferax
volcanii. J Proteome Res 7: 5033–5039.Klussmann S, Franke P, Bergmann U, Kostka S & Wittmann-
Liebold B (1993) N-terminal modification and amino-acid
sequence of the ribosomal protein HmaS7 from Haloarcula
marismortui and homology studies to other ribosomal
proteins. Biol Chem Hoppe Seyler 374: 305–312.Koch MK & Oesterhelt D (2005) MpcT is the transducer for
membrane potential changes in Halobacterium salinarum.
Mol Microbiol 55: 1681–1694.Koch MK, Staudinger WF, Siedler F & Oesterhelt D (2008)
Physiological sites of deamidation and methyl esterification
in sensory transducers of Halobacterium salinarum. J Mol
Biol 380: 285–302.Konrad Z & Eichler J (2002) Lipid modification of proteins
in Archaea: attachment of a mevalonic acid-based lipid
moiety to the surface-layer glycoprotein of Haloferax
volcanii follows protein translocation. Biochem J 366: 959–964.
Koretke KK, Lupas AN, Warren PV, Rosenberg M & Brown JR
(2000) Evolution of two-component signal transduction.
Mol Biol Evol 17: 1956–1970.Kuntz C, Sonnenbichler J, Sonnenbichler I, Sumper M &
Zeitler R (1997) Isolation and characterization of dolichol-
linked oligosaccharides from Haloferax volcanii. Glycobiology
7: 897–904.Lanyi JK (2004) Bacteriorhodopsin. Annu Rev Physiol 66: 665–
688.
LaRonde-LeBlanc N & Wlodawer A (2005a) A family portrait
of the RIO kinases. J Biol Chem 280: 37297–37300.LaRonde-LeBlanc N & Wlodawer A (2005b) The RIO kinases:
an atypical protein kinase family required for ribosome
biogenesis and cell cycle progression. Biochim Biophys Acta
1754: 14–24.Lechner J & Sumper M (1987) The primary structure of a
procaryotic glycoprotein. Cloning and sequencing of the cell
surface glycoprotein gene of Halobacteria. J Biol Chem 262:
9724–9729.Lechner J & Sumper M (1989) Structure and biosynthesis
of prokaryotic glycoproteins. Annu Rev Biochem 58:
173–194.Lechner J & Wieland F (1989) Structure and biosynthesis of
prokaryotic glycoproteins. Annu Rev Biochem 58: 173–194.Lechner J, Wieland F & Sumper M (1985a) Biosynthesis of
sulfated saccharides N-glycosidically linked to the protein
via glucose. Purification and identification of sulfated
dolichyl monophosphoryl tetrasaccharides from
Halobacteria. J Biol Chem 260: 860–866.
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Rev 37 (2013) 583–606Published by John Wiley & Sons Ltd. All rights reserved
602 J. Eichler & J. Maupin-Furlow
Lechner J, Wieland F & Sumper M (1985b) Transient
methylation of dolichyl oligosaccharides is an obligatory
step in halobacterial sulfated glycoprotein biosynthesis.
J Biol Chem 260: 8984–8989.Lee DY, Teyssier C, Strahl BD & Stallcup MR (2005) Role of
protein methylation in regulation of transcription. Endocr
Rev 26: 147–170.Lee HS, Kim YJ, Bae SS, Jeon JH, Lim JK, Jeong BC, Kang SG
& Lee JH (2006) Cloning, expression, and characterization
of a methionyl aminopeptidase from a hyperthermophilic
archaeon Thermococcus sp. NA1. Mar Biotechnol 8: 425–432.Leonard CJ, Aravind L & Koonin EV (1998) Novel families of
putative protein kinases in bacteria and archaea: evolution
of the “eukaryotic” protein kinase superfamily. Genome Res
8: 1038–1047.Lindbeck JC, Goulbourne EA Jr, Johnson MS & Taylor BL
(1995) Aerotaxis in Halobacterium salinarium is
methylation-dependent. Microbiology 141: 2945–2953.Lowther WT & Matthews BW (2000) Structure and function
of the methionine aminopeptidases. Biochim Biophys Acta
1477: 157–167.Lowther WT & Matthews BW (2002) Metalloaminopeptidases:
common functional themes in disparate structural
surroundings. Chem Rev 102: 4581–4608.Lupas A, Flanagan JM, Tamura T & Baumeister W (1997)
Self-compartmentalizing proteases. Trends Biochem Sci 22:
399–404.Macek B, Gnad F, Soufi B, Kumar C, Olsen JV, Mijakovic I &
Mann M (2008) Phosphoproteome analysis of E. coli reveals
evolutionary conservation of bacterial Ser/Thr/Tyr
phosphorylation. Mol Cell Proteomics 7: 299–307.Mackay DT, Botting CH, Taylor GL & White MF (2007) An
acetylase with relaxed specificity catalyses protein
N-terminal acetylation in Sulfolobus solfataricus. Mol
Microbiol 64: 1540–1548.Magidovich H, Yurist-Doutsch S, Konrad Z, Ventura VV,
Hitchen PG, Dell A & Eichler J (2010) AglP is a S-adenosyl-
L-methionine-dependent methyltransferase that participates
in the N-glycosylation pathway of Haloferax volcanii. Mol
Microbiol 76: 190–199.Mann RK & Beachy PA (2004) Novel lipid modifications of
secreted protein signals. Annu Rev Biochem 73: 891–923.Mao DY, Neculai D, Downey M et al. (2008) Atomic
structure of the KEOPS complex: an ancient protein
kinase-containing molecular machine. Mol Cell 32:
259–275.Marquez V, Frohlich T, Armache JP et al. (2011) Proteomic
characterization of archaeal ribosomes reveals the presence
of novel archaeal-specific ribosomal proteins. J Mol Biol 405:
1215–1232.Marsh VL, Peak-Chew SY & Bell SD (2005) Sir2 and the
acetyltransferase, Pat, regulate the archaeal chromatin
protein, Alba. J Biol Chem 280: 21122–21128.Marsh VL, McGeoch AT & Bell SD (2006) Influence of
chromatin and single strand binding proteins on the activity
of an archaeal MCM. J Mol Biol 357: 1345–1350.
Martinez A, Traverso JA, Valot B, Ferro M, Espagne C,
Ephritikhine G, Zivy M, Gigilone C & Meinnel T (2008)
Extent of N-terminal modifications in cytosolic proteins
from eukaryotes. Proteomics 8: 2809–2831.Matsumoto S, Igura M, Nyirenda J, Matsumoto M, Yuzawa S,
Noda NN, Inagaki F & Kohda D (2012) Crystal structure of
the C-terminal globular domain of oligosaccharyltransferase
from Archaeoglobus fulgidus at 1.75 �A resolution.
Biochemistry 51: 4157–4166.Mattar S, Scharf B, Kent SB, Rodewald K, Oesterhelt D &
Engelhard M (1994) The primary structure of halocyanin,
an archaeal blue copper protein, predicts a lipid anchor for
membrane fixation. J Biol Chem 269: 14939–14945.Maupin-Furlow J (2012) Proteasomes and protein conjugation
across domains of life. Nat Rev Microbiol 10: 100–111.Maupin-Furlow JA, Gil MA, Humbard MA, Kirkland PA,
Li W, Reuter CJ & Wright AJ (2005) Archaeal proteasomes
and other regulatory proteases. Curr Opin Microbiol 8:
720–728.Maupin-Furlow JA, Humbard MA & Kirkland PA (2012)
Extreme challenges and advances in archaeal proteomics.
Curr Opin Microbiol 15: 351–356.Mazel D, Pochet S & Marliere P (1994) Genetic
characterization of polypeptide deformylase, a distinctive
enzyme of eubacterial translation. EMBO J 13: 914–923.Meinnel T, Peynot P & Giglione C (2005) Processed N-termini
of mature proteins in higher eukaryotes and their
major contribution to dynamic proteomics. Biochimie 87:
701–712.Meinnel T, Serero A & Giglione C (2006) Impact of the
N-terminal amino acid on targeted protein degradation. Biol
Chem 387: 839–851.Mengele R & Sumper M (1992) Drastic differences in
glycosylation of related S-layer glycoproteins from moderate
and extreme halophiles. J Biol Chem 267: 8182–8185.Mescher MF & Strominger JL (1976) Purification and
characterization of a prokaryotic glucoprotein from the cell
envelope of Halobacterium salinarium. J Biol Chem 251:
2005–2014.Mescher MF & Strominger JL (1978) Glycosylation of the
surface glycoprotein of Halobacterium salinarium via a
cyclic pathway of lipid-linked intermediates. FEBS Lett 89:
37–41.Meyer BH, Zolghadr B, Peyfoon E et al. (2011) Sulfoquinovose
synthase - an important enzyme in the N-glycosylation
pathway of Sulfolobus acidocaldarius. Mol Microbiol 82:
1150–1163.Milligan DL & Koshland DE Jr (1990) The amino terminus of
the aspartate chemoreceptor is formylmethionine. J Biol
Chem 265: 4455–4460.Min KT, Hilditch CM, Diederich B, Errington J & Yudkin MD
(1993) Sigma F, the first compartment-specific transcription
factor of B. subtilis, is regulated by an anti-sigma factor that
is also a protein kinase. Cell 74: 735–742.Miranda HV, Nembhard N, Su D, Hepowit N, Krause DJ,
Pritz JR, Phillips C, Soll D & Maupin-Furlow JA (2011)
FEMS Microbiol Rev 37 (2013) 583–606 ª 2012 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved
Protein modification in the haloarchaea 603
E1- and ubiquitin-like proteins provide a direct link
between protein conjugation and sulfur transfer in archaea.
P Natl Acad Sci USA 108: 4417–4422.Muff TJ & Ordal GW (2007) Assays for CheC, FliY & CheX as
representatives of response regulator phosphatases. Methods
Enzymol 423: 336–348.Nadolski MJ & Linder ME (2007) Protein lipidation. FEBS
J 274: 5202–5210.Naor A, Thiaville PC, Altman-Price N, Cohen-Or I, Allers T,
Crecy-Lagard V & Gophna U (2012) A genetic investigation
of the KEOPS complex in halophilic archaea. PLoS ONE 7:
e43013.
Ng WV, Kennedy SP, Mahairas GG et al. (2000) Genome
sequence of Halobacterium species NRC-1. P Natl Acad Sci
USA 97: 12176–12181.Ng SY, Chaban B, VanDyke DJ & Jarrell KF (2007) Archaeal
signal peptidases. Microbiology 153: 305–314.Nordmann B, Lebert MR, Alam M, Nitz S, Kollmannsberger
H, Oesterhelt D & Hazelbauer GL (1994) Identification of
volatile forms of methyl groups released by Halobacterium
salinarium. J Biol Chem 269: 16449–16454.Nunoura T, Takaki Y, Kakuta J et al. (2011) Insights into the
evolution of Archaea and eukaryotic protein modifier
systems revealed by the genome of a novel archaeal group.
Nucleic Acids Res 39: 3204–3223.Nutsch T, Oesterhelt D, Gilles ED & Marwan W (2005) A
quantitative model of the switch cycle of an archaeal
flagellar motor and its sensory control. Biophys J 89: 2307–2323.
Oesterhelt D & Stoeckenius W (1971) Rhodopsin-like protein
from the purple membrane of Halobacterium halobium. Nat
New Biol 233: 149–152.Oke M, Carter LG, Johnson KA et al. (2010) The Scottish
Structural Proteomics Facility: targets, methods and outputs.
J Struct Funct Genomics 11: 167–180.Oren A, Ginzburg M, Ginzburg BZ, Hochstein LI & Volcani
BE (1990) Haloarcula marismortui (Volcani) sp. nov., nom.
rev., an extremely halophilic bacterium from the Dead Sea.
Int J Syst Bacteriol 40: 209–210.Oxenrider KA & Kennelly PJ (1993) A protein-serine
phosphatase from the halophilic archaeon Haloferax
volcanii. Biochem Biophys Res Commun 194: 1330–1335.Paik WK, Paik DC & Kim S (2007) Historical review: the
field of protein methylation. Trends Biochem Sci 32:
146–152.Park MH, Cooper HL & Folk JE (1981) Identification of
hypusine, an unusual amino acid, in a protein from human
lymphocytes and of spermidine as its biosynthetic precursor.
P Natl Acad Sci USA 78: 2869–2873.Park MH, Nishimura K, Zanelli CF & Valentini SR (2010)
Functional significance of eIF5A and its hypusine
modification in eukaryotes. Amino Acids 38: 491–500.Paulus H (2000) Protein splicing and related forms of protein
autoprocessing. Annu Rev Biochem 69: 447–496.Pechlivanis M & Kuhlmann J (2006) Hydrophobic
modifications of Ras proteins by isoprenoid groups and
fatty acids – more than just membrane anchoring. Biochim
Biophys Acta 1764: 1914–1931.Perazzona B & Spudich JL (1999) Identification of methylation
sites and effects of phototaxis stimuli on transducer
methylation in Halobacterium salinarum. J Bacteriol 181:
5676–5683.Pfeiffer F, Schuster SC, Broicher A, Falb M, Palm P, Rodewald
K, Ruepp A, Soppa J, Tittor J & Oesterhelt D (2008)
Evolution in the laboratory: the genome of Halobacterium
salinarum strain R1 compared to that of strain NRC-1.
Genomics 91: 335–346.Pickl A, Johnsen U & Schonheit P (2012) Fructose degradation
in the haloarchaeon Haloferax volcanii involves bacterial
type phosphoenolpyruvate-dependent phosphotransferase
system, fructose-1-phosphate kinase and Class II fructose-
1,6-bisphosphate aldolase. J Bacteriol, 194: 3088–3097.Pohlschroder M, Gimenez MI & Jarrell KF (2005) Protein
transport in Archaea: sec and twin arginine translocation
pathways. Curr Opin Microbiol 8: 713–719.Ponting CP, Aravind L, Schultz J, Bork P & Koonin EV (1999)
Eukaryotic signalling domain homologues in archaea and
bacteria. Ancient ancestry and horizontal gene transfer.
J Mol Biol 289: 729–745.Porter SL, Wadhams GH & Armitage JP (2011) Signal
processing in complex chemotaxis pathways. Nat Rev
Microbiol 9: 153–165.Ramesh V & RajBhandary UL (2001) Importance of the
anticodon sequence in the aminoacylation of tRNAs by
methionyl-tRNA synthetase and by valyl-tRNA synthetase in
an Archaebacterium. J Biol Chem 276: 3660–3665.Ranjan N, Damberger FF, Sutter M, Allain FH & Weber-Ban E
(2010) Solution structure and activation mechanism of
ubiquitin-like small archaeal modifier proteins. J Mol Biol
405: 1040–1055.Reuter CJ, Kaczowka SJ & Maupin-Furlow JA (2004)
Differential regulation of the PanA and PanB proteasome-
activating nucleotidase and 20S proteasomal proteins of
the haloarchaeon Haloferax volcanii. J Bacteriol 186: 7763–7772.
Roderick SL & Matthews BW (1993) Structure of the cobalt-
dependent methionine aminopeptidase from Escherichia coli:
a new type of proteolytic enzyme. Biochemistry 32: 3907–3912.
Rudolph J & Oesterhelt D (1995) Chemotaxis and phototaxis
require a CheA histidine kinase in the archaeon
Halobacterium salinarium. EMBO J 14: 667–673.Rudolph J, Tolliday N, Schmitt C, Schuster SC & Oesterhelt D
(1995) Phosphorylation in halobacterial signal transduction.
EMBO J 14: 4249–4257.Sagami H, Kikuchi A & Ogura K (1994) Novel isoprenoid
modified proteins in Halobacteria. Biochem Biophys Res
Commun 203: 972–978.Sagami H, Kikuchi A & Ogura K (1995) A novel type of
protein modification by isoprenoid-derived materials.
Diphytanylglycerylated proteins in Halobacteria. J Biol Chem
270: 14851–14854.
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Rev 37 (2013) 583–606Published by John Wiley & Sons Ltd. All rights reserved
604 J. Eichler & J. Maupin-Furlow
Saini P, Eyler DE, Green R & Dever TE (2009) Hypusine-
containing protein eIF5A promotes translation elongation.
Nature 459: 118–121.Schumann H & Klink F (1989) Archaebacterial protein
contains hypusine, a unique amino-acid characteristic for
eukaryotic translation initiation factor-4D. Syst Appl
Microbiol 11: 103–107.Shi L, Potts M & Kennelly PJ (1998) The serine, threonine
and/or tyrosine-specific protein kinases and protein
phosphatases of prokaryotic organisms: a family portrait.
FEMS Microbiol Rev 22: 229–253.Shi L, Bischoff KM & Kennelly PJ (1999) The icfG gene cluster
of Synechocystis sp. strain PCC 6803 encodes an Rsb/Spo-
like protein kinase, protein phosphatase and two
phosphoproteins. J Bacteriol 181: 4761–4767.Shigi N (2012) Post-translational modification of cellular
proteins by a ubiquitin-like protein in bacteria. J Biol Chem
287: 17568–17577.Shiio Y & Eisenman RN (2003) Histone sumoylation is
associated with transcriptional repression. P Natl Acad Sci
USA 100: 13225–13230.Sinensky M (2000) Recent advances in the study of prenylated
proteins. Biochim Biophys Acta 1484: 93–106.Solbiati J, Chapman-Smith A, Miller JL, Miller CG & Cronan
JE Jr (1999) Processing of the N termini of nascent
polypeptide chains requires deformylation prior to
methionine removal. J Mol Biol 290: 607–614.Soppa J (2006) From genomes to function: haloarchaea as
model organisms. Microbiology 152: 585–590.Soppa J (2010) Protein acetylation in archaea, bacteria &
eukaryotes. Archaea 2010: 820681.
Spudich JL & Stoeckenius W (1980) Light-regulated retinal-
dependent reversible phosphorylation of Halobacterium
proteins. J Biol Chem 255: 5501–5503.Srinivasan M, Mehta P, Yu Y, Prugar E, Koonin EV, Karzai
AW & Sternglanz R (2011) The highly conserved KEOPS/
EKC complex is essential for a universal tRNA modification,
t6A. EMBO J 30: 873–881.Starai VJ & Escalante-Semerena JC (2004) Identification of the
protein acetyltransferase (Pat) enzyme that acetylates acetyl-
CoA synthetase in Salmonella enterica. J Mol Biol 340: 1005–1012.
Storch KF, Rudolph J & Oesterhelt D (1999) Car: a cytoplasmic
sensor responsible for arginine chemotaxis in the archaeon
Halobacterium salinarum. EMBO J 18: 1146–1158.Storf S, Pfeiffer F, Dilks K, Chen ZQ, Imam S & Pohlschr€oder
M (2010) Mutational and bioinformatic analysis of
haloarchaeal lipobox-containing proteins. Archaea 2010:
410975.
Streif S, Oesterhelt D & Marwan W (2010) A predictive
computational model of the kinetic mechanism of stimulus-
induced transducer methylation and feedback regulation
through CheY in archaeal phototaxis and chemotaxis. BMC
Syst Biol 4: 27.
Strom MS, Nunn DN & Lory S (1993) A single bifunctional
enzyme, PilD, catalyzes cleavage and N-methylation of
proteins belonging to the type IV pilin family. P Natl Acad
Sci USA 90: 2404–2408.Sumper M, Berg E, Mengele R & Strobel I (1990) Primary
structure and glycosylation of the S-layer protein of
Haloferax volcanii. J Bacteriol 172: 7111–7118.Sundberg SA, Alam M, Lebert M, Spudich JL, Oesterhelt D &
Hazelbauer GL (1990) Characterization of Halobacterium
halobium mutants defective in taxis. J Bacteriol 172: 2328–2335.
Szabo Z, Stahl AO, Albers SV, Kissinger JC, Driessen AJM &
Pohlschroder M (2007) Identification of diverse archaeal
proteins with class III signal peptides cleaved by distinct
archaeal prepilin peptidases. J Bacteriol 189: 772–778.Szymanski CM & Wren BW (2005) Protein glycosylation
in bacterial mucosal pathogens. Nat Rev Microbiol 3:
225–237.Tahirov TH, Oki H, Tsukihara T, Ogasahara K, Yutani K,
Ogata K, Izu Y, Tsunasawa S & Kato I (1998) Crystal
structure of methionine aminopeptidase from
hyperthermophile, Pyrococcus furiosus. J Mol Biol 284: 101–124.
Thao S & Escalante-Semerena JC (2011) Control of protein
function by reversible Ne-lysine acetylation in bacteria. Curr
Opin Microbiol 14: 200–204.Tran HJ, Allen MD, L€owe J & Bycroft M (2003) Structure of
the Jab1/MPN domain and its implications for proteasome
function. Biochemistry 42: 11460–11465.Tripepi M, Imam S & Pohlschroder M (2010) Haloferax
volcanii flagella are required for motility but are not
involved in PibD-dependent surface adhesion. J Bacteriol
192: 3093–3102.Tripepi M, You J, Temel S, Onder O, Brisson D &
Pohlschr€oder M (2012) N-glycosylation of Haloferax volcanii
flagellins requires known Agl proteins and is essential for
biosynthesis of stable flagella. J Bacteriol 194: 4876–4887.Tsunasawa S, Izu Y, Miyagi M & Kato I (1997) Methionine
aminopeptidase from the hyperthermophilic Archaeon
Pyrococcus furiosus: molecular cloning and overexpression
in Escherichia coli of the gene and characteristics of the
enzyme. J Biochem 122: 843–850.Tyagi N, Anamika K & Srinivasan N (2010) A framework for
classification of prokaryotic protein kinases. PLoS ONE 5:
e10608.
Uthandi S, Saad B, Humbard MA & Maupin-Furlow JA (2010)
LccA, an archaeal laccase secreted as a highly-stable
glycoprotein into the extracellular medium of Haloferax
volcanii. Appl Environ Microbiol 76: 733–743.VanDyke DJ, Wu J, Logan SM, Kelly JF, Mizuno S, Aizawa SI
& Jarrell KF (2009) Identification of genes involved in the
assembly and attachment of a novel flagellin N-linked
tetrasaccharide important for motility in the archaeon
Methanococcus maripaludis. Mol Microbiol 72: 633–644.van Valkenburgh C, Chen X, Mullins C, Fang H & Green N
(1999) The catalytic mechanism of endoplasmic reticulum
signal peptidase appears to be distinct from most eubacterial
signal peptidases. J Biol Chem 274: 11519–11525.
FEMS Microbiol Rev 37 (2013) 583–606 ª 2012 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved
Protein modification in the haloarchaea 605
Varshavsky A (2011) The N-end rule pathway and regulation
by proteolysis. Protein Sci 20: 1298–1345.Verma R, Aravind L, Oania R, McDonald WH, Yates JR III,
Koonin EV & Deshaies RJ (2002) Role of Rpn11
metalloprotease in deubiquitination and degradation by the
26S proteasome. Science 298: 611–615.Vetting MW, S de Carvalho LP, Yu M, Hegde SS, Magnet S,
Roderick SL & Blanchard JS (2005) Structure and functions
of the GNAT superfamily of acetyltransferases. Arch Biochem
Biophys 433: 212–226.Wagner S & Klug G (2007) An archaeal protein with homology
to the eukaryotic translation initiation factor 5A shows
ribonucleolytic activity. J Biol Chem 282: 13966–13976.Wang F, Liu M, Qiu R & Ji C (2011) The dual role of
ubiquitin-like protein Urm1 as a protein modifier and
sulfur carrier. Protein Cell 2: 612–619.Wardleworth BN, Russell RJ, Bell SD, Taylor GL & White MF
(2002) Structure of Alba: an archaeal chromatin protein
modulated by acetylation. EMBO J 21: 4654–4662.Weerapana E & Imperiali B (2006) Asparagine-linked protein
glycosylation: from eukaryotic to prokaryotic systems.
Glycobiology 16: 91R–101R.Wieland F, Dompert W, Bernhardt G & Sumper M (1980)
Halobacterial glycoprotein saccharides contain covalently
linked sulphate. FEBS Lett 120: 110–114.Wieland F, Heitzer R & Schaefer W (1983) Asparaginylglucose:
novel type of carbohydrate linkage. P Natl Acad Sci USA 80:
5470–5474.Wieland F, Paul G & Sumper M (1985) Halobacterial
flagellins are sulfated glycoproteins. J Biol Chem 260:
15180–15185.Wilson HL, Aldrich HC & Maupin-Furlow J (1999) Halophilic
20S proteasomes of the archaeon Haloferax volcanii:
purification, characterization and gene sequence analysis.
J Bacteriol 181: 5814–5824.Wu J, Ohta N, Zhao JL & Newton A (1999) A novel bacterial
tyrosine kinase essential for cell division and differentiation.
P Natl Acad Sci USA 96: 13068–13073.Wurgler-Murphy SM, King DM & Kennelly PJ (2004) The
Phosphorylation Site Database: a guide to the serine-,
threonine- and/or tyrosine-phosphorylated proteins in
prokaryotic organisms. Proteomics 4: 1562–1570.
YaDeau JT, Klein C & Blobel G (1991) Yeast signal peptidase
contains a glycoprotein and the Sec11 gene product. P Natl
Acad Sci USA 88: 517–521.Yang X, Kang CM, Brody MS & Price CW (1996) Opposing
pairs of serine protein kinases and phosphatases transmit
signals of environmental stress to activate a bacterial
transcription factor. Genes Dev 10: 2265–2275.Yao T & Cohen RE (2002) A cryptic protease couples
deubiquitination and degradation by the proteasome.
Nature 419: 403–407.Yurist-Doutsch S, Abu-Qarn M, Battaglia F, Morris HR,
Hitchen PG, Dell A & Eichler J (2008) aglF, aglG and aglI,
novel members of a gene cluster involved in the
7N-glycosylation of the Haloferax volcanii S-layer
glycoprotein. Mol Microbiol 69: 1234–1245.Yurist-Doutsch S, Magidovich H, Ventura VV, Hitchen PG,
Dell A & Eichler J (2010) N-glycosylation in Archaea: on
the coordinated actions of Haloferax volcanii AglF and
AglM. Mol Microbiol 75: 1047–1058.Zhang F, Paterson AJ, Huang P, Wang K & Kudlow JE (2007)
Metabolic control of proteasome function. Physiology 22:
373–379.Zhao K, Chai X & Marmorstein R (2003) Structure of a Sir2
substrate, Alba, reveals a mechanism for deacetylation-
induced enhancement of DNA binding. J Biol Chem 278:
26071–26077.Zhou G, Kowalczyk D, Humbard MA, Rohatgi S & Maupin-
Furlow JA (2008) Proteasomal components required for cell
growth and stress responses in the haloarchaeon Haloferax
volcanii. J Bacteriol 190: 8096–8105.Zhu BC, Drake RR, Schweingruber H & Laine RA (1995)
Inhibition of glycosylation by amphomycin and sugar
nucleotide analogs PP36 and PP55 indicates that
Haloferax volcanii beta-glucosylates both glycoproteins and
glycolipids through lipid-linked sugar intermediates:
evidence for three novel glycoproteins and a novel
sulfated dihexosyl-archaeol glycolipid. Arch Biochem
Biophys 319: 355–364.Zhu W, Reich CI, Olsen GJ, Giometti CS & Yates JR III
(2004) Shotgun proteomics of Methanococcus jannaschii
and insights into methanogenesis. J Proteome Res 3: 538–548.
ª 2012 Federation of European Microbiological Societies FEMS Microbiol Rev 37 (2013) 583–606Published by John Wiley & Sons Ltd. All rights reserved
606 J. Eichler & J. Maupin-Furlow