vipp1: a very important protein in plastids?!

Journal of Experimental Botany, Vol. 63, No. 4, pp. 1699–1712, 2012doi:10.1093/jxb/err357 Advance Access publication 29 November, 2011

REVIEW PAPER

Vipp1: a very important protein in plastids?!

Ute C. Vothknecht1,*, Stephanie Otters1, Raoul Hennig2 and Dirk Schneider2,*

1 Department of Biology I, LMU Munich, D-82152 Planegg-Martinsried, Germany2 Department of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, D-55128 Mainz, Germany

* To whom correspondence should be addressed. E-mail: [email protected] or [email protected]

Received 5 September 2011; Revised 13 October 2011; Accepted 14 October 2011

Abstract

As a key feature in oxygenic photosynthesis, thylakoid membranes play an essential role in the physiology of plants,

algae, and cyanobacteria. Despite their importance in the process of oxygenic photosynthesis, their biogenesis has

remained a mystery to the present day. A decade ago, vesicle-inducing protein in plastids 1 (Vipp1) was described to

be involved in thylakoid membrane formation in chloroplasts and cyanobacteria. Most follow-up studies clearly

linked Vipp1 to membranes and Vipp1 interactions as well as the defects observed after Vipp1 depletion in

chloroplasts and cyanobacteria indicate that Vipp1 directly binds to membranes, locally stabilizes bilayer structures,and thereby retains membrane integrity. Here current knowledge about the structure and function of Vipp1 is

summarized with a special focus on its relationship to the bacterial phage shock protein A (PspA), as both proteins

share a common origin and appear to have retained many similarities in structure and function.

Key words: Chloroplasts, cyanobacteria, PspA, thylakoid membrane biogenesis, vesicle transport, Vipp1.

Emergence of Vipp1 as a factor for thylakoidbiogenesis

Thylakoids are characteristic structures of cyanobacteria

and chloroplasts, and the major complexes of oxygenic

photosynthesis are embedded in these membranes. The

thylakoid membrane system evolved in cyanobacteria in

close correlation with the evolution of oxygenic photosyn-

thesis and generated a novel bacterial compartment, the

thylakoid lumen. Thylakoids are exclusively linked toorganisms carrying out oxygenic photosynthesis, and the

facility to build up and to modify such a complex mem-

brane system has been transferred from the cyanobacterial

ancestor to the plant cell during the endosymbiotic estab-

lishment of chloroplasts (reviewed in Vothknecht and

Westhoff, 2001). Thylakoid membrane biogenesis is a multi-

dimensional process, involving a concerted interplay of lipid,

protein, and pigment synthesis, as well as their transport andintegration into functional membrane-integral complexes. It

is still unclear whether they assemble completely de novo or

whether their membrane scaffold derives from the inner

envelope of chloroplasts or the plasma membrane of

cyanobacteria, respectively. While insertion of photosyn-

thetic proteins into already existing thylakoid lipid bilayers

has been analysed in some detail (reviewed in Aldridge

et al., 2009), there is very little information about compo-

nents involved in the formation of the thylakoid membrane

itself (reviewed in Vothknecht and Westhoff, 2001; Nickel-

sen et al., 2010).The vesicle-inducing protein in plastids 1 (Vipp1) was first

described in 1994 as a protein located in the chloroplasts of

Pisum sativum (Li et al., 1994). Vipp1 is nuclear encoded

and contains an N-terminal sequence sharing common

characteristics with other chloroplast-directed transit pep-

tides. Thus, like most plastidal proteins, Vipp1 is translated

as a precursor on cytosolic ribosomes and is afterwards

imported into the chloroplast by the TOC–TIC generalimport pathway (Schnell et al., 1997). An initial analysis

further suggested that the protein is located at the inner

envelope membrane as well as the thylakoids. Due to its

localization and its mature size of 30 kDa, the protein was

Abbreviations: PG, phosphatidyl glycerol; PS, photosystem; Psp, phage shock protein; Sec, general secretory pathway; Tat, twin-arginine translocation pathway; TIC,translocon on the inner chloroplast membrane; TOC, translocon on the outer chloroplast membrane; Vipp1, vesicle-inducing protein in plastids 1.ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

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first named M30 (membrane-associated protein of 30 kDa)

(Li et al., 1994). However, Vipp1 does not display any

features of intrinsic membrane proteins, as was revealed

by structural analysis and supported by experimental

approaches (Li et al., 1994; Kroll et al., 2001). Instead,

further studies demonstrated a rather peripheral association

of Vipp1 with chloroplast membranes. The unique localiza-

tion of Vipp1 at the two membranes bordering the stromalcompartment led to the presumption that Vipp1 might

somehow be involved in the assembly of the thylakoid

membrane system (Li et al., 1994). More precisely, it has

been suggested that the protein is involved in the transport

of lipids, which are synthesized at the chloroplast inner

envelope. A potential involvement of Vipp1 in thylakoid

membrane biogenesis was supported several years later by

the drastic phenotype of an Arabidopsis thaliana highchlorophyll fluorescence mutant line (hcf155), in which the

expression of the vipp1 gene was strongly reduced due to

a T-DNA insertion in its promoter region (Kroll et al.,

2001). Mutant plants expressed only about a fifth of the

Vipp1 wild-type protein content under normal growth

conditions and were incapable of autotrophic growth. Even

on sugar-supplemented growth medium they showed

a strong albinotic phenotype and did not survive beyondthe stage of a small rosette (Kroll et al., 2001). Transmission

electron microscopy of leaf sections revealed that mutant

plants were not able to build a properly structured

thylakoid membrane system. Their chloroplasts, while

almost normal in size, contained very few internal mem-

branes in rather severely distorted structures that are

diffusely distributed inside the organelle. Correlating with

their lack of thylakoid membranes, the capacity foroxygenic photosynthesis was extremely decreased (Kroll

et al., 2001). Furthermore, hcf155 mutant plants displayed

another phenotype in accordance with Vipp1’s alleged

function in lipid transfer. Formation of vesicular structures

at the inner envelope of chloroplasts could no longer be

observed in the mutants, indicating that Vipp1 might be

essential for this feature. Thus, the protein was renamed

‘vesicle-inducing protein in plastids 1’ (Kroll et al., 2001).At the same time, a vipp1 mutant of Synechocystis sp. PCC

6803 was described. While not fully segregated, mutant cells

had a drastically reduced Vipp1 content. This cyanobacte-

rial mutant displayed a similar phenotype to the hcf155

mutant line, showing a comparable loss of thylakoid

membrane content and structure, as well as reduced photo-

synthetic activity (Westphal et al., 2001a). These findings not

only supported the function of Vipp1 independently in twodifferent organisms but also indicated a phylogenetically

conserved function.

PspA and Vipp1, a shared past (andpresent?)

Phylogenetic analyses strongly support Vipp1 as a protein

of prokaryotic origin that is found exclusively in cyanobac-

teria and chloroplasts (Li et al., 1994; Kroll et al., 2001;

Westphal et al., 2001a; Fuhrmann et al., 2009a). This

indicates a development in parallel to the emergence of the

thylakoid membrane system and a connection to the

evolution of oxygenic photosynthesis. However, proteins

with significant similarities to Vipp1 have been identified in

several non-photosynthetic bacteria (Fig. 1) in form of the

phage shock protein A (PspA) (Brissette et al., 1990). PspA

was first characterized following infection of Escherichia coli

cells with filamentous phages, whereby the phage gene pIV

protein was found to induce pspA expression to extremely

high levels (Brissette et al., 1990). It was subsequently

shown that PspA is a member of a bacterial stress response

system, referred to as the ‘phage shock protein system’ (Psp

system) (Brissette et al., 1991). The Psp system in E. coli

responds not only to the invasion of filamentous phages but

also to a number of different extracytoplasmic stressfactors, such as heat, hyperosmotic conditions, organic

solvents (Brissette et al., 1991; Weiner and Model, 1994;

Kobayashi et al., 1998), inhibition of lipid biosynthesis

(Bergler et al., 1994), and mislocalization or blockage of the

protein export machinery (Kleerebezem and Tommassen,

1993; Kleerebezem et al., 1996; DeLisa et al., 2004). A

common denominator of these stress conditions is the

reduction of the proton motive force across the cytoplasmicmembrane. In total, the Psp system in E. coli comprises

seven psp genes. In addition to the pspABCDE operon, the

transcriptional activator PspF as well as the effector protein

PspG are encoded by separate genes (Brissette et al., 1991;

Elderkin et al., 2002; Lloyd et al., 2004). Furthermore, the

E. coli two-component system consisting of ArcA and ArcB

has been shown to be involved in signal sensing and

induction of the Psp-mediated stress response (Jovanovicet al., 2006, 2009).

PspA is a hydrophilic protein of 25 kDa that, like Vipp1,

shares no features of integral membrane proteins but

appears to be peripherally associated with the cytoplasmic

membrane of bacterial cells (Brissette et al., 1990; Cserzo

et al., 1997; Kobayashi et al., 2007). As the most abundant

protein and primary effector of the Psp response, PspA is

responsible for several phenotypes connected to this system(Weiner and Model, 1994; Kleerebezem et al., 1996; DeLisa

et al., 2004). It is assumed that PspA supports the bacterial

cell in stress situations by blocking proton leakage

(Kobayashi et al., 2007) or by inducing the down-regulation

of proton motive force-consuming processes (Engl et al.,

2009). The Psp system appears to be widespread throughout

all bacterial domains. Psp proteins have been identified in

Gram-negative and Gram-positive bacteria, as well as inarchaea and cyanobacteria. In Yersinia enterocolitica, where

the stress response is well established, the Psp system was

found to be induced by two outer membrane secretins.

Furthermore, it plays an important role in maintaining the

membrane stability of the pathogen during cell infection

(Darwin and Miller, 2001). Psp proteins are also highly

expressed in Salmonella enterica (Eriksson et al., 2003) and

Shigella flexneri (Lucchini et al., 2005) during macrophageinfection. A functional analysis of the Psp response in the

halophilite archaeon Halifax volcanii has pointed out that

1700 | Vothknecht et al.


Fig. 1. Phylogenetic tree construction of PspA and Vipp1. Included in the analysis were proteins with sequenced similarity to PspA and

Vipp1 from Actinomyces odontolyticus (Aco), Alteromonas macleodii (Alm), Anabaena variabilis (Anv1, Anv2), Arabidopsis thaliana (Ath),

Arthrobacter chlorophenolicus (Arc), Azospirillum sp. (Azs), Bacillus sp. (Bas), Bacillus subtilis (Bacs), Bifidobacterium animalis (Bia),

Bifidobacterium gallicum (Big), Burkholderia phymatum (Bup), Caldicellulosiruptor bescii (Cab), Caldicellulosiruptor owensensis (Cao),

Candidatus Korarchaeum cryptofilum (Cac), Chlamydomonas reinhardtii (Chr), Chlorella variabilis (Chv), Chloroherpeton thalassium (Cht),

Citrobacter sp. (Cis), Clostridium botulinum (Clb), Clostridium sp. (Cls), Crocosphaera watsonii (Crw1, Crw2), Cyanothece sp. (Cys1,

Cys2), Delftia sp. (Des), Desulfatibacillum alkenivorans (Desa), Desulfobacterium autotrophicum (Dea), Desulvovibrio vulgaris RCH1 (Dev),

Desulvovibrio vulgaris DP4 (Desv), Ectocarpus siliculosus (Ecs), Eikenella corrodens (Eic), Enterobacter sp. (Ens), Erythrobacter sp. (Ers),

Escherichia coli (Esc), Gloeobacter violaceus (Glv), Gluconobacter oxydans (Glo), Glycine max (Glm), Haliangium ochraceum (Hao),

Haloarcula marismortui (Ham), Haloquadratum walsbyi (Haw), Halorhabdus utahensis (Hau), Haloterrigena turkmenica (Hat), Helio-

bacterium modesticaldum (Hem), Hirschia baltica (Hib), Hordeum vulgare (Hov), Hypomonas neptunicum (Hyn), Klebsiella pneumoniae

(Klp), Kytococcus sedentarius (Kys), Lyngbya majuscule (Lym1, Lym2), Lyngbya sp. (Lys1, Lys2), Micrococcus luteus (Mil), Microcoleus

vaginatus (Miv1, Miv2) Micromonas pusilla (Mip), Micromonas spec. (Mis), Myxococcus xanthus (Myx), Natrialba magadii (Nam), Neisseria

subflava (Nes), Nodularia spumigena (Nods1, Nods2), Nostoc punctiforme (Nop1, Nop2), Nostoc sp. (Nos1, Nos2) Novosphingobium

sp. (Novs) Oryza sativa (Ors), Oscillatoria sp. (Oss1, Oss2), Phaeodactylum tricornutum (Pht), Photobacterium profundum (Phop),

Phyllostachys edulis (Phe), Physcomitrella patens (Php), Picea sitchensis (Pics), Pisum sativum (Pis), Planctomyces brasiliensis (Plb),

Planctomyces maris (Plm), Plesiocystis pacifica SIR-1 (Plp, Plep) Populus trichocarpa (Pot), Prochlorococcus marinus (Prm), Proteus

mirabilis (Prs), Pseudomonas syringae (Pss), Renibacterium salmoninarum (Res), Rhodospirillum rubrum (Rhr), Ricinus communis (Ric),

Ruminococcus sp. (Rus), Salmonella enterica (Sae), Selaginella moellendorffii (Smo), Shewanella baltica (Shb), Sorangium cellulosum

(Soc), Sorghum bicolour (Sob), Sphingobium japonicum (Spj), Streptomyces lividans (Stl), Streptomyces pristinaespiralis (Stp),

Vipp1: a very import protein in plastids | 1701


pspA expression was up-regulated in response to salt stress

(Bidle et al., 2008). Phylogenetic analysis indicates that

a minimal functional Psp system—containing just PspA, B,

C, and F—is conserved in a-, d- and c-proteobacteria,whereas other Psp components show much less conserva-

tion or are even absent (Darwin, 2005). Moreover, PspA

homologues also appear in organisms without connection

to any other component of the Psp response, suggestingeither a strong reduction of the Psp system or a newly

acquired function of the remaining PspA homologue

(reviewed in Darwin, 2005). In Streptomyces lividans,

expression of pspa is strongly induced under membrane

stress (Vrancken et al., 2008), which indicates that PspA has

retained an important effector function in the stress re-

sponse of this Gram-positive bacterium despite the loss of

other Psp members. LiaH, the PspA homologue of theGram-positive bacterium Bacillus subtilis (Mascher et al.,

2004), belongs to the Lia system that is conserved in all

Firmicutes, Bacillus, and Listeria species, where it regulates

the bacterial stress response. The regulatory Lia system is

comprised of the LiaRS two-component system linked to

LiaF, a membrane-anchored negative regulator of LiaR-

dependent gene expression. The LiaRS two-component

system recognizes different antibiotics and triggers a 100-to 1000-fold induction of the liaIHGFSR operon. Beside

a strong induction in response to external cell wall anti-

biotics that affect the lipid II cycle (Mascher et al., 2004;

Pietiainen et al., 2005), expression of the Lia system is also

induced by alkaline shock, as well as by treatment with

organic solvents and secretion stress (Petersohn et al., 2001;

Mascher et al., 2004; Hyyrylainen et al., 2005). The Lia

system of B. subtilis thus seems to represent a complexregulatory stress response system in Gram-positive bacteria

comparable with the Psp response system found in E. coli

(Wolf et al., 2010). While most components of the Psp and

Lia system differ, the primary target proteins of both

systems, LiaH and PspA, show sequence and structural

homology, indicating a similar mode of action.

Many cyanobacterial genomes possess two genes encod-

ing proteins with significant homology to members of thePspA/Vipp1 protein family (Fig. 1), even if they lack other

genes encoding proteins of the original Psp response. Based

on a phylogenetic analysis, these proteins divide into two

different clades, suggesting that cyanobacteria require PspA

as well as Vipp1. In the genome of Anabaena sp. PCC 7120,

the genes encoding these two proteins are localized adjacent

to each other with a gap of only 162 bp between the stop

codon of the former and the start codon of the latter(Kaneko et al., 2001). A similar arrangement can be found

in other cyanobacteria, suggesting that Vipp1 has evolved in

an ancestor of today’s cyanobacteria from PspA by gene

duplication and was passed to the plant lineage by the

cyanobacterial endosymbiont (Westphal et al., 2001a). No

pspA gene has been identified explicitly in any plant or alga,

indicating that PspA was either absent in the original

endosymbiont or was lost in the plant lineage shortly after

the endosymbiotic event. While cyanobacterial PspA pro-

teins cluster together with the Vipp1 branch containing bothcyanobacterial and plant Vipp1 proteins, PspA proteins

from bacteria other than cyanobacteria cluster in a separate

clade, which also includes PspA proteins from Archaea

(Fig. 1). This strongly supports the hypothesis that Vipp1

evolved from a cyanobacterial PspA rather than originating

in a different bacterial ancestor.

Initially, the situation appeared rather straightforward,

with non-photosynthetic bacteria possessing only PspA,chloroplasts only Vipp1, and cyanobacteria encoding both

proteins (Fig. 1). This observation linked the presence of

Vipp1 to thylakoid membranes and supported a thylakoid-

specific function. Such an assumption was further sup-

ported by the apparent lack of a vipp1 gene in the genome

of Gloeobacter violoceus sp. PCC 7421 (Nakamura et al.,

2003), a unicellular, obligate photoautotrophic bacterium

that branches at an early stage of cyanobacterial evolution(Nelissen et al., 1995; Honda et al., 1999). It contains

structural features that differ from those of common

cyanobacteria, such as the lack of thylakoid membranes,

and its photosynthetic machinery is located on the cytoplas-

mic membrane (Rippka et al., 1974). As Gloeobacter

provides several primordial characteristics, the organism is

an important tool to investigate the origin of oxygenic

photosynthesis (Nakamura et al., 2003; Mimuro et al.,2008). In agreement with the alleged function of Vipp1 in

thylakoid biogenesis, its absence in Gloeobacter provided

a simple explanation for the inability of this organism to

build up a functional thylakoid membrane system. Gloeo-

bacter does contain a protein of the Vipp1/PspA family,

which is annotated as a PspA homologue due to certain

conserved structural characteristics (see below). However, in

phylogenetic analysis, this Gloeobacter protein is placed ona separate side branch of the cyanobacterial Vipp1/PspA

clade, so that a definite phylogenetic relationship to either

PspA or Vipp1 cannot be obtained (Fig. 1). The availability

of many more completely sequenced cyanobacterial

genomes has caused the situation to become more complex.

The genomes of several cyanobacteria, such as some

Prochlorococcus species, also lack a gene encoding Vipp1,

despite the presence of thylakoids in these organisms(Dufresne et al., 2003). In some of these cyanobacteria, the

supposed lack of Vipp1 might be due to a misannotation of

Synechococcus sp. (Syns1, Syns2), Synechocystis sp. (Sys1, Sys2), Thalassiosira pseudonana (Thp), Thiomonas intermedia (Thi),

Trichodesmium erythraeum (Tre1a, Tre1b, Tre2), Vibrio sp. (Vibs), Vitis viniferum (Viv), Volvox carteri (Voc), Yersinia pestis (Yep), and

Yersinia enterocolitica (Yee). Sequence alignments were obtained by Clustal 1.8 (Thompson et al., 1997). Phylogenetic tree construction

was performed by maximum likelihood using Phyml 3.0 (Guindon and Gascuel, 2003). Transit sequences were omitted from the plant

sequences for the analysis.



the vipp1 genes as pspA, but in some of them Vipp1 indeed

appears to be absent. While these findings question the

direct correlation of Vipp1 to thylakoid membrane evolu-

tion, it is possible that these organisms have secondarily lost

Vipp1 and now utilize a different system for thylakoid

membrane biogenesis and/or maintenance.

Since Vipp1 originated from PspA, the question arose as

to what extent PspA function is still conserved in Vipp1proteins. Expression of Vipp1 from the cyanobacterium

Synechocystis sp. PCC6803 in an E. coli DpspA deletion

strain prevented blockage of protein translocation (DeLisa

et al., 2004), indicating that the cyanobacterial Vipp1 could

somehow functionally replace the bacterial PspA. In

contrast, endogenous PspA present in a Synechocystis

Dvipp1 depletion strain was not sufficient to compensate

the deficiency causing the drastic phenotype of thosemutants, and thus to take over the function of Vipp1

(Westphal et al., 2001a; Fuhrmann et al., 2009b; Gao and

Xu, 2009). Obviously, PspA is not necessary for the

bacterial cells in the absence of stress factors, since E. coli

DpspA mutants are perfectly viable (Brissette et al., 1991).

In contrast, vipp1 mutants from Arabidopsis and Synecho-

cystis show a dramatic phenotype even if vipp1 expression is

not completely abolished but merely strongly decreased(Kroll et al., 2001; Westphal et al., 2001a; Aseeva et al.,

2007; Gao and Xu, 2009). Along with the fact that many

cyanobacteria still possess both proteins, there is strong

evidence that Vipp1 proteins have gained a novel function

in cyanobacteria and chloroplasts. However, Vipp1 has also

been clearly correlated to stress response. In Synechocystis,

Vipp1 was found amongst those plasma membrane proteins

exhibiting the highest enhancements under high salt con-ditions (Huang et al., 2006). In contrast, the vipp1 gene was

significantly down-regulated in the cold (Suzuki et al.,

2001). Also, the Chlamydomonas Vipp1 was found in

a group of proteins whose expression was up-regulated

under high light (Im and Grossman, 2002). Nevertheless, in

all these cases it is not clear whether the change in vipp1

expression represents a specific stress response or whether it

is due to a general function of Vipp1 in thylakoidmembrane synthesis and maintenance, which is affected

under these conditions.

The structure of Vipp1

Vipp1 proteins of plants and algae exhibit an N-terminal

extension that has evolved as a transit peptide to ensure the

import of the cytosolic-translated protein into the chloro-

plast (Li et al., 1994; Westphal et al., 2001a). Since the

transit peptide is removed after translocation, it has no

bearing on the structure of the mature protein and is

therefore mostly excluded in structural comparisons be-tween Vipp1 and PspA. While the phylogenetic relationship

between Vipp1 and PspA is evident, it remains unclear to

what extent the function of Vipp1 was altered in the course

of evolution and which changes within the protein might

affect any difference in function between the two proteins.

PspA and Vipp1 proteins display certain conservation in

primary amino acid sequence but, even in the same

organism, such as in Synechocystis sp. PCC 6803, they share

just ;30% sequence identity and 50% sequence similarity

(Bultema et al., 2010). Nevertheless, all PspA/Vipp1 family

members exhibit a common N-terminal core structure, the

so-called PspA domain that is ;220 amino acids in length

(Westphal et al., 2001a). Furthermore, the secondary andtertiary structures as well as the intrinsic propensity of PspA

and Vipp1 to form higher ordered oligomers appear to be

highly conserved (Aseeva et al., 2004; Hankamer et al., 2004;

Fuhrmann et al., 2009a; Bultema et al., 2010).

Based on computational predictions and spectroscopic

measurements, an essentially exclusively a-helical structurewas identified for the PspA-like domain of Vipp1/PspA

proteins (Fig. 2A) (Hankamer et al., 2004; Fuhrmann et al.,2009a; Bultema et al., 2010). Only few amino acids within

this region are predicted to be unstructured and to interrupt

the helical domain. In contrast to PspA, Vipp1 proteins are

longer due to a random coil spacer of variable size,

connecting the PspA-like domain to a short a-helicaldomain of ;30 amino acids (Westphal et al., 2001a). This

defined a-helical extension at the C-terminus appears to be

a characteristic feature of Vipp1 proteins that clearlydistinguishes Vipp1 from PspA.

The a-helical regions of the PspA domain have a high

propensity to form coiled-coil structures and to homo-

oligomerize to higher ordered rings with molecular masses

>1 MDa. While the exact tertiary structure of Vipp1 is

presently unknown, large parts of the coiled-coil domain are

Fig. 2. Vipp1 oligomer formation. (A) Schematic of the Synecho-

cystis Vipp1 protein sequence and the positions of the helical

domains (HDs) according to regions defined for E. coli PspA

(Elderkin et al., 2005). Putative functions of the individual domains

in oligomerization are indicated. Amino acid residues 222–267

represent the C-terminal extension typical for Vipp1 proteins.

Predicted a-helical regions are indicated by grey bars.

(B) Formation of Vipp1 rings from Vipp1 monomers involves

intermediate dimeric, tetrameric, and string-like states.

(C) Individual Vipp1 rings eventually interact to form double rings

and rod-like structures.



involved in oligomerization (Aseeva et al., 2004; Bultema

et al., 2010). Based on structural analysis, PspA of E. coli

was subdivided into four helical domains HD1–HD4 and, in

a fragmentation approach, oligomerization of truncated E.

coli PspA proteins was analysed in detail (Elderkin et al.,

2005; Joly et al., 2009) (Fig. 2A). The centre regions HD2

and HD3 alone support formation of a PspA hexamer,

whereas the HD1–HD2–HD3 protein forms dimers. Thus,while HD2 and HD3 are critical for hexamer formation,

HD3 negatively influences hexamerization, thereby however

stabilizing a dimeric PspA structure. HD4 supports forma-

tion of a PspA 36mer ring structure, and, thus, this domain

appears to connect individual dimers. It might be assumed

that similar regions control assembly and disassembly of

Vipp1 oligomers in vivo, as indicated in Fig. 2A, but this has

not been assessed in detail yet. Accessibility of the individualhelical regions and their involvement in defined intermono-

mer contacts might be the key for controlling the actual

oligomeric state of PspA/Vipp1 proteins. Chaperones might

further be crucially involved in shielding defined regions of

the protein for controlled Vipp1 assembly/disassembly (as

discussed below).

While the tertiary structures of PspA and Vipp1 are not

well defined, the quaternary structures of both have beenanalysed to a certain degree in recent years. Both proteins

form large homo-oligomeric rings, albeit with different

dimensions (Aseeva et al., 2004; Hankamer et al., 2004; Liu

et al., 2007; Fuhrmann et al., 2009a; Bultema et al., 2010).

PspA from E. coli forms a single, distinct ring structure with

a 9-fold rotational symmetry and a calculated mass of

;1 MDa. It has been suggested that this ring assembles

from nine tetrameric building blocks (Hankamer et al.,2004). The PspA ring has an outer diameter of 20 nm, an

inner diameter of 9.5 nm, and a height of ;8.5 nm

(Hankamer et al., 2004). In contrast, Vipp1 proteins of

Arabidopsis and Synechocystis organize into variable ring

structures with molecular masses of up to ;2 MDa (Aseeva

et al., 2004; Fuhrmann et al., 2009a). While for Synechocys-

tis Vipp1 at least six different ring structures with a 12–17

internal rotational symmetry and outer diameters between25 nm and 33 nm have been identified, all rings have

a uniform height of 22 nm (Fuhrmann et al., 2009a). Based

on recent results it seems likely that multiple copies of

monomers (or smaller oligomeric building blocks) assemble

into string-like structures representing intermediates during

ring assembly or disassembly. Such an assumption is

supported by the observation that some string-like struc-

tures have indeed been observed in the case of theSynechocystis protein. Furthermore, controlled disassembly

of Vipp1 results in formation of distinct tetrameric and

dimeric (dis)assembly intermediates, indicating that Vipp1

dimers are the minimal building blocks of the Vipp1 rings

(Fuhrmann et al., 2009a). At higher concentrations, in-

dividual Vipp1 rings eventually stack together and form

barrel-like double-ring structures, as observed with Vipp1

from cyanobacteria and higher plants (Aseeva et al., 2004;Fuhrmann et al., 2009a). Such double-ring structures

potentially represent an assembly intermediate during

formation of multiple-ring stacking structures with a rod-

like shape, as detected after heterologous expression and in

vitro refolding of the Chlamydomonas reinhardtii Vipp1 (Liu

et al., 2007). Such scaffold-like structures have also been

described for PspA (Standar et al., 2008). Figure 2B and C

summarize all presently identified Vipp1 structures and

a potential assembly/disassembly pathway.

The characteristic C-terminal extension of Vipp1 proteinsdoes not appear to be essential for the formation of rings or

rods (Aseeva et al., 2004; Liu et al., 2007). Fusing a protein

to the Vipp1 C-terminus does not interfere either with its

oligomerization propensity or with thylakoid biogenesis in

Arabidopsis (Aseeva et al., 2007). Thus, the C-terminus is

probably not crucially involved in formation of Vipp1

oligomers and a free C-terminus is not essential for Vipp1

activity. However, as the C-terminal extension appears to bea characteristic feature of Vipp1, it is crucial for discrimi-

nating PspA from the PspA-like Vipp1 proteins.

Based on a sequence alignment of Vipp1 homologues of

cyanobacteria, algae, and higher plants, the following

consensus sequences of the Vipp1 C-terminus was gener-

ated: V/I/L-D/E-X-E-L-E/D-E/D-L-R/(K)-R/K-X-L/I-D/

N-X-(L)-X3 (Fig. 3A). Even though the amino acids are

not strictly conserved at most positions, the properties ofindividual amino acid side chains are preserved at various

places. The Vipp1 C-terminus is predicted to form an

amphiphilic a-helix with one non-polar, hydrophobic face,

containing two acidic residues at the N-terminus and two

basic residues at the C-terminus (Fig. 3B). As the

C-terminus most probably protrudes out of the ring

structures (Aseeva et al., 2004), these conserved features of

the Vipp1 C-terminal helix eventually allow and controldefined interactions with proteins, lipids, or membranes.

Structural dynamics, interactions, andfunctions

Most data somehow link both PspA and Vipp1 to mem-

branes. In chloroplasts, Vipp1 is associated with the inner

envelope as well as with thylakoid membranes, and in

cyanobacteria the protein localizes in close vicinity to both

the cytoplasmic and thylakoid membranes (Srivastava et al.,

2005; Fuhrmann et al., 2009a). Detailed studies on membrane

attachment revealed that the PspA protein of E. coli is able tobind directly to membrane lipids, having a clear preference for

lipids with negatively charged head groups (Kobayashi et al.,

2007). In vivo, however, PspA is mainly recruited to mem-

branes by the membrane-integral proteins PspB and C, and it

has been suggested that these proteins sense stress signals and

modulate PspF activity by a controlled interaction with PspA

(Joly et al., 2010). Only upon release of PspA from the PspBC

complex the interaction of PspA with the transcriptionactivator PspF permitted. This releases the suppression of the

psp operon by PspF and results in overexpression of PspA.

Subsequently, PspA binds to membranes directly for its

function in stress protection.



Different oligomeric structures of PspA appear to have

different functions in E. coli. When interacting with PspF,PspA is present as a hexamer, whereas formation of the

PspA 36mer appears to be required for direct membrane

attachment and maintenance of membrane integrity. In

contrast to PspA, no hexameric Vipp1 structure has been

reported, and, as Vipp1 does not seem to interact with any

component similar to PspF, a hexameric assembly stage

might not exist. Extraction of Vipp1 from cyanobacterial

thylakoids furthermore suggests that the protein binds asrings to membranes (Fuhrmann et al., 2009a). Soluble

Vipp1 rings have been identified in the cyanobacterial

cytoplasm, which might be less stable than membrane-

bound rings (Fuhrmann et al., 2009a). While for PspA

membrane attachment appears to be regulated by the

oligomeric state, it is not clear how this is regulated for

Vipp1. It is noteworthy that in cyanobacteria and plants, two

Vipp1 forms showing an ;2 kDa difference in apparentmolecular mass have been identified (Kroll et al., 2001;

Aseeva et al., 2004; Liu et al., 2005; Fuhrmann et al., 2009a).

While the difference between these two Vipp1 forms has not

been determined yet, Vipp1 might be post-translationally

modified. Thus, the two Vipp1 forms observed on SDS–gels

might represent two structurally different Vipp1 populations

having different membrane binding affinities.Individual Vipp1 monomers have an intrinsic propensity

to homo-oligomerize and to aggregate. In an Arabidopsis

mutant strain containing a massively decreased Vipp1

content, oligomers have not been observed, and thus it

seems that a critical Vipp1 concentration is needed for

complex formation (Aseeva et al., 2007). At higher concen-

trations, rod-like structures form, which have been sug-

gested to represent a chloroplastic microtubule-like system(Liu et al., 2007). Rod-like structures of unknown origin

have indeed been observed in chloroplasts as well as in

cyanobacteria (Anderson et al., 1973; Lawrence et al., 1984;

van de Meene et al., 2006), and it is therefore intriguing to

speculate that such cytoskeleton-like elements could be used

as tracks for a directed transport of proteins and/or vesicles

to distinct locations within chloroplasts or cyanobacteria.

Nevertheless, while Vipp1 rings stack at higher concentra-tions, no rod structures have been reported thus far for

PspA proteins. Formation of rods, which are essentially

identical to Vipp1 rods, is also observed in vitro by the ring-

forming, membrane-integral Holine protein of bacterio-

phage k (Savva et al., 2008). Since artificial formation of

Fig. 3. Consensus sequence of the Vipp1 C-terminus. (A) Alignment of Vipp1 sequences from cyanobacteria, green algea, brown algae,

and plants revealed a conserved consensus sequence within the last 44 amino acids, forming the a-helical Vipp1 C-terminus. The

degree of conservation is shown by the number of bits (Crooks et al., 2004). The approach disclosed a strict conservation of side chain

properties in several positions, even if the amino acids were not conserved. (B) Helical wheel projection of the consensus sequence

underlined in A predicts the formation of an amphiphatic a-helix with a hydrophilic face on one side of the helix (indicated). Furthermore,

the helix is bookended by two conserved acidic residues at the N-terminus and two basic residues at the C-terminus. Amino acid

properties are indicated: magenta, acidic residue; blue, basic residue; black/grey, non-polar residue.



the identified rod structures here strongly depended on the

experimental conditions, the existence of Vipp1 rods in vivo

still has to be proven unambiguously.

Oligomerization and membrane attachment of Vipp1

might be controlled by a chaperone-mediated disassembly

of Vipp1 rings. Shielding defined regions of the PspA

domain would allow controlled assembly/disassembly of

Vipp1 rings (compare Fig. 2A) and consequently membranebinding of the protein. Vipp1 of the green alga C. reinhardtii

interacts with the chloroplast chaperone Hsp70B, as well as

with the Hsp40 homologue CDJ2 (Liu et al., 2005). Binding

of these chaperones to Vipp1 resulted in disassembly of

large Vipp1 rings and rods in vitro (Liu et al., 2007),

although it is not yet clear whether the rings disassemble

into monomers or to lower ordered oligomeric states. Thus,

the Hsp70/Hsp40 system might be part of a cellular systemwhich controls assembly/disassembly of Vipp1 oligomers

and thereby its membrane attachment. Binding to chloroplast

chaperones might also be an early step in Vipp1 import into

the organelle, to prevent unspecific aggregation of the protein

and to control oligomer formation (Fig. 4). Such an

assumption is supported by the recent observation that Vipp1

can be co-precipitated together with components of the TIC–

TOC protein import machinery of pea chloroplasts (Jouhetand Gray, 2009). While a single Hsp70 and Hsp40 protein has

been identified in the stroma of Chlamydomonas chloroplasts

(Liu et al., 2005), multiple Hsp70/DnaK and Hsp40/DnaJ

proteins are encoded in cyanobacteria (Nimura et al., 2001;

Rupprecht et al., 2007, 2010; Sato et al., 2007; Duppre et al.,

2011). It is thus not clear yet which DnaK/DnaJ pair might

regulate assembly/disassembly of Vipp1 rings in cyanobac-

teria. However, involvement of a special cyanobacterial

DnaK and DnaJ protein in thylakoid membrane biogenesis

has been suggested (Nimura et al., 1996; Oguchi et al., 1997),

and thus it is intriguing to speculate that these proteins

interact with Vipp1. Such an assumption is supported by the

observation that the specified DnaK and DnaJ chaperones

are not encoded in the genome of the thylakoid-free

cyanobacterium Gloeobacter (Nakamura et al., 2003).

Hsp90C has also been reported to form a complex withHsp70, Hsp40, and Vipp1 in Clamydomonas chloroplasts

(Heide et al., 2009). It is noteworthy that an Arabidopsis

hsp90C mutant line has defects in chloroplast development

and thylakoid stacking (Cao et al., 2003). The point mutation

in Hsp90C in this mutant might affect interaction of

chloroplast Hsp90 with Vipp1 (Heide et al., 2009), which

subsequently perturbs thylakoid membrane biogenesis.

Involvement of Vipp1 in the integration of thylakoidmembrane proteins has been suggested based on the

observation of a direct interaction of Vipp1 with the

Albino3.2 protein in Chlamydomonas chloroplasts (Gohre

et al., 2006). The thylakoid-localized Albino3.2 belongs to

the conserved YidC/Oxa1p/Alb3 protein family, whose

members function as insertases, chaperones, and/or assembly

factors for transmembrane proteins (reviewed in Wang and

Dalbey, 2010). It has been suggested that Vipp1 is eitherinvolved in membrane integration of Albino3.2 or that Vipp1

delivers substrates to Albino3.2 for membrane integration

(Gohre et al., 2006). Albino3.2 has an essential role during

assembly of photosystem II (PSII) and PSI reaction centres

in thylakoid membranes (Gohre et al., 2006). During

Albino3.2-mediated membrane integration and/or assembly

of protein complexes, the membrane structure might be

locally perturbed and Vipp1 could eventually be required tostabilize the membrane. In Chlamydomonas, depletion of the

Albino3.2 protein results in overproduction of Vipp1 (Gohre

et al., 2006), while depletion of YidC in E. coli causes an

increased expression of PspA (van der Laan et al., 2003).

Thus, Vipp1 might be involved in stabilizing the membrane

structure during Albino3.2-mediated protein insertion but

also participates in maintaining the membrane integrity after

Albino3.2 depletion. Vipp1 (additionally?) could associatewith the TIC complex to stabilize the inner envelope

membrane in the vicinity of the TIC complex during TIC-

mediated protein integration. Involvement of PspA-like

proteins in protein translocation/integration is further sup-

ported by the observation that increased PspA expression has

been observed in E. coli cells having a defect in the Sec

pathway (Kleerebezem and Tommassen, 1993). Furthermore,

overexpression of PspA in S. lividans improved the functionof both the Sec and Tat pathway (Vrancken et al., 2007).

PspA has been suggested to minimize proton leakage of the

bacterial plasma membrane during Tat-mediated protein

translocation/integration, and thus the occurrence of Vipp1

and/or PspA has been linked to the Tat pathway of bacteria,

archaea, and plant chloroplasts (DeLisa et al., 2004). In-

terestingly, this function can be taken over by the full-length

Vipp1 as well as by a C-terminally truncated Vipp1 ofSynechocystis PCC 6803 in an E. coli pspA deletion strain

(DeLisa et al., 2004).

Fig. 4. Import and assembly of Vipp1 in chloroplasts. Pre-Vipp1 is

imported from the cytoplasm via the translocons at the outer and

inner envelope of chloroplasts (TOC and TIC) into the chloroplast

stroma. Molecular chaperones prevent misfolding and unspecific

aggregation. Oligomerization of Vipp1 is guided by the Hsp70 and

Hsp40 chaperones in an ATP-dependent manner. Oligomeric

Vipp1 rings eventually interact with the inner envelope (IE) or

thylakoid membranes (TM).



Thylakoid membranes, the photosyntheticelectron transfer chain, and Vipp1

An increased amount of thylakoid membranes and chlor-

ophylls correlates with increased light harvesting, and

chloroplasts adapt to high-light growth conditions by re-

duction of thylakoid membranes (Anderson et al., 1973;

Lichtenthaler et al., 1981; Melis and Harvey, 1981). Further-more, in both chloroplasts and cyanobacteria, the ratio of

the two photosystems, PSI and PSII, alters in response to

changing light conditions (Chow et al., 1990; Kim et al.,

1993; Murakami et al., 1997). A comparative proteomic

analysis of bundle sheet and mesophyll chloroplasts in maize

has shown that mesophyll cell plastids, which contain a larger

thylakoid membrane system, have a 10-fold increased Vipp1

content (Majeran et al., 2005). Mesophyll cells also containhigh amounts of PSII, which is mostly lacking in bundle

sheet chloroplasts. Depletion of Vipp1 in both higher plants

and cyanobacteria resulted in a massive decrease of assem-

bled thylakoid membrane layers and in defects in the

photosynthetic electron transfer chain (Kroll et al., 2001;

Westphal et al., 2001a). In Arabidopsis the defect could not

be attributed to a deficiency of a single photosynthetic

complex, but appeared to be caused by dysfunction of theentire photosynthetic electron transfer chain (Kroll et al.,

2001). Vipp1-depleted plants displayed a dramatic reduction

of both photosystems as well as of the cytochrome b6f

complex. A disturbed energy transfer between antenna and

photosynthetic core complexes has also been noticed, and the

light-harvesting complexes are not properly attached to the

photosystems (Aseeva et al., 2007).

Electron microscopic analysis of Synechocystis Dvipp1mutant cells showed a similar reduction in thylakoid

membranes. Furthermore, the overall thylakoid membrane

morphology was disturbed and the thylakoid membranes

were less well arranged than in the wild-type strain

(Fuhrmann et al., 2009b). This observation indicates that

the composition of the thylakoid membranes and/or thyla-

koid membrane modelling is affected by Vipp1 depletion. In

cyanobacteria, the two photosystems typically form dimersand trimers, respectively, and trimerization of PSI was

affected by Vipp1 depletion (Fuhrmann et al., 2009b).

Furthermore, the relative PSI:PSII ratio was also signifi-

cantly decreased in the depletion strain, which indicates an

impaired activity of the photosynthetic electron transfer

chain (Schneider et al., 2001, 2004; Volkmer et al., 2007).

After controlled depletion of Vipp1 in Synechocystis a dras-

tic effect on photosynthetic complexes has been observed(Gao and Xu, 2009), which further suggests a direct effect

of Vipp1 on photosystem biogenesis, assembly, or function.

Vipp1 and the need for vesicles inchloroplasts and cyanobacteria

The debate on how thylakoid membranes are formed in

mature chloroplasts without a connection to the inner

envelope has been longstanding (reviewed in Vothknecht

and Westhoff, 2001). Several of the thylakoid constituents,

such as monogalactosidyl diacylglycerol and other lipids,

are synthesized within the inner envelope (Douce, 1974;

Joyard and Stumpf, 1981; Kobayashi et al., 2007). Conse-

quently, there is a need for regulated transport between

internal membranes within chloroplasts. This might be

achieved by a vesicle transport system of eukaryotic origin

that was suggested to exist in chloroplasts of plants(reviewed in von Wettstein, 2001). Vesicle formation can be

observed in chloroplasts of higher plants under certain

experimental conditions (Westphal et al., 2001b) but is no

longer detected in the Arabidopsis Vipp1 depletion strain

(Kroll et al., 2001). It is indeed conceivable that Vipp1 of

higher plants might support thylakoid biogenesis and

maintenance by sustaining vesicle traffic and thereby

ensuring the flow of lipids and other non-lipid componentsfrom the inner envelope to the thylakoids.

It has long been debated whether thylakoid membranes

also represent a completely independent internal membrane

system in cyanobacteria or whether they are still linked to

the cytoplasmic membrane. However, recent structural

analyses strongly suggest that thylakoids indeed are not

directly connected to cyanobacterial cytoplasmic mem-

branes, and thus represent a separate internal membraneentity (Liberton et al., 2006; van de Meene et al., 2006;

Nevo et al., 2007; Schneider et al., 2007). Furthermore, it

has been suggested that precursors of the cyanobacterial

photosystems are pre-assembled within the plasma mem-

brane (Zak et al., 2001; Keren et al., 2005), implying the

existence of a distinct lipid and protein transfer system from

the cytoplasmic to the thylakoid membrane. Such transfer

might involve vesicles or a temporary connection of themembrane systems. Thylakoid centres in cyanobacteria may

be the origin of thylakoids, as these regions appear to

connect thylakoid membranes with the cytoplasmic mem-

brane in some cyanobacteria (Nickelsen et al., 2010).

However, thylakoid centre structures are not typical and

highly conserved in these organisms, indicating that they are

not generally involved in thylakoid membrane biogenesis.

Vesicle accumulation was detected in chloroplasts ofA. thaliana and P. sativum by transmission electron

microscopy while providing conditions that are known to

prevent vesicle fusion in the cytosol. These include

a decrease in the temperature or treatment with toxins

such as ophiobolin A and microcystin, established inhib-

itors of cytosolic proteins involved in this process (Morre

et al., 1991; Kroll et al., 2001; Westphal et al., 2001b).

Until now, no vesicle transport system has been observedunder those conditions in cyanobacteria, green algae, or

even charophytes, implying that the specific vesicle trans-

port system of chloroplasts might be restricted to

embryophytes (bryophytes, pteridophytes, and spermato-

phytes) and has evolved somewhere in an ancestor

between today’s charyophytes and bryophytes (Westphal

et al., 2003). Nevertheless, this has been a point of

ongoing debate, with different experimental evidencesupporting the occurrence of vesicle transport in photo-

synthetic organisms other than embryophytes (Hoober



et al., 1991). After staining the cyanobacterial cytoplas-

mic membrane with a fluorescent dye, intracellular

vesicular structures appeared within individual cells

(Schneider et al., 2007). Also, in the cyanobacterium

Microcoleus sp., large vesicular structures have been

identified by electron microscopy (Nevo et al., 2007).

Vipp1 might have a function similar to coat proteins in

the secretory pathway, which stabilize highly bent mem-branes during vesicle budding. For E. coli PspA it has been

observed that it predominantly binds to the cell poles of the

bacterium, where the membranes are highly bent and where

membrane stress and proton leakage might be higher than

in planar bilayer regions (Engl et al., 2009). Thus, Vipp1

might be involved in stabilizing highly bent membranes, as

is the case during vesicle formation. The diverse oligomeric

Vipp1 ring structures could also be involved in vesicleconstriction, similar to dynamins in eukaryotes (Fuhrmann

et al., 2009a). While assembly and disassembly of dynamin

is regulated by its GTPase domain, controlled disassembly

and formation of smaller Vipp1 rings eventually involves

Hsp70 and Hsp40, as discussed above. PspA and Vipp1

show homology to the eukaryotic Snf7 protein, which is

involved in protein sorting and transport from eukaryotic

endosomes to the vacuole/lysosome, and all three proteinsare clustered in a single protein family in the pfam database

(Finn et al., 2009). Furthermore, proteins with homologies to

components of the vesicular transfer system of the secretory

pathway have been predicted to localize to chloroplasts, and

some of the predicted proteins also have clear homologues in

cyanobacteria (Andersson and Sandelius, 2004). Thus, it

cannot be excluded per se that a vesicular transfer system

exists in chloroplasts and cyanobacteria.

Vipp1 function: does a common themeemerge?

All studies carried out in chloroplasts and cyanobacteria

indicate that Vipp1 binds directly to membranes, locally

stabilizes bilayer structures, and thereby retains membrane

integrity. While initial observations indicated an involve-

ment of Vipp1 in vesicle formation in chloroplasts, the

observed perturbations in an Arabidopsis vipp1 depletion

strain might be caused by an altered membrane structure

and/or composition. However, also during vesicle forma-tion, membranes deform and Vipp1 might have a function

in stabilizing bent membranes, similar to coat proteins.

Involvement of Vipp1 in vesicle transfer between internal

membrane systems in chloroplasts and cyanobacteria is still

an appealing hypothesis as a general function of Vipp1,

since a membrane chaperone role could most probably also

be fulfilled by PspA, which is still present in cyanobacteria.

Nevertheless, Vipp1 has some distinct features differentfrom PspA. While both PspA and Vipp1 form ring

structures, the ring dimensions are dramatically different

and this leads to the necessity for variance in the PspA and

Vipp1 tertiary structure. PspA monomers most probably

form a kinked helix bundle, whereas Vipp1 may have a long

stretched a-helical shape (Bultema et al., 2010). These

different quaternary structures of PspA and Vipp1 eventu-

ally determine the exact physiological functions of the

individual proteins. A key to the specific function of Vipp1

could also be the C-terminal extension unique to Vipp1

proteins. This extension appears to correlate with the

appearance of thylakoid membranes, as Gloeobacter con-

tains neither Vipp1 nor thylakoid membranes, whereas itencodes a PspA protein, which could be crucially involved

in maintaining the integrity of the cytoplasmic membrane.

It is noteworthy that in the genome of Gloeobacter, genes

involved in sulphoquinovosyl diacylglycerol synthesis are

also missing (Nakamura et al., 2003). Sulphoquinovosyl

diacylglycerol is a constituent of typical thylakoid mem-

branes, and, like phosphatidyl glycerol (PG), contains

a negatively charged head group. Depletion of Vipp1 inSynechocystis results in a decreased PSI content and in

a reduced trimerization propensity of PSI (Fuhrmann et al.,

2009b), a phenotype also observed after PG depletion

(Hagio et al., 2000; Sato et al., 2000). Also in Arabidopsis

PG is required for photosynthesis, and a mutant having

inactivated PG synthesis was no longer able to grow

photoautotrophically and showed a severe reduction in

thylakoid membrane (Hagio et al., 2002). As PspA fromE. coli has been shown to interact preferentially with the

head groups of negatively charged lipids (Kobayashi et al.,

2007), all these observations link PspA and Vipp1 not only

generally to membranes, but more specifically to negatively

charged membrane lipids. Defined interaction of Vipp1 with

negatively charged lipids might thus enable spatial and

temporal control of Vipp1’s action on membrane systems in

cyanobacteria and chloroplasts.However, all in all the exact function of both PspA and

Vipp1 remain elusive. Yet considering the importance of

Vipp1 for the viability of cyanobacteria and plants, it will

be essential in the future to obtain a more conclusive picture

of the role of this proteins in membrane biogenesis and

maintenance.

Acknowledgements

This work was supported by grants from the Deutsche

Forschungsgemeinschaft to UCV (SFB-TR1, project A6)

and DS (FOR 929, SCHN 690/3-1) and from the Center of

Complex Matter (COMATT) to DS. RH is supported by

a fellowship of the Max Planck Graduate Center Mainz.

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vipp1: a very important protein in plastids?!

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