proteome analysis of aspergillus fumigatus total membrane ... · sphingosine-1-phosphate (s1p)...
Post on 19-Oct-2020
0 Views
Preview:
TRANSCRIPT
RESEARCH
Proteome Analysis of Aspergillus fumigatus Total MembraneProteins Identifies Proteins Associated with the Glycoconjugatesand Cell Wall Biosynthesis Using 2D LC-MS/MS
Haomiao Ouyang • Yuanming Luo •
Lei Zhang • Yanjie Li • Cheng Jin
� Springer Science+Business Media, LLC 2009
Abstract We attempted to identify membrane proteins
associated with the glycoconjugates and cell wall biosyn-
thesis in the total membrane preparations of Aspergillus
fumigatus. The total membrane preparations were first run
on 1D gels, and then the stained gels were cut and submitted
to in-gel digestion followed by 2D LC-MS/MS and database
search. A total of 530 proteins were identified with at least
two peptides detected with MS/MS spectra. Seventeen
integral membrane proteins were involved in N-, O-glyco-
sylation or GPI anchor biosynthesis. Nine membrane pro-
teins were involved in cell wall biosynthesis. Eight proteins
were identified as enzymes involved in sphingolipid syn-
thesis. In addition, the proteins involved in cell wall and
ergosterol biosynthesis can potentially be used as antifungal
drug targets. Our method, for the first time, clearly provided
a global view of the membrane proteins associated with
glycoconjugates and cell wall biosynthesis in the total
membrane proteome of A. fumigatus.
Keywords Aspergillus fumigatus �Cell wall biosynthesis � Glycoconjugate �Membrane protein � Mass spectrometry
Introduction
Aspergillus fumigatus is the most common mold pathogen
of humans and causes both invasive disease in immuno-
compromised patients and allergic disease in patients with
atopic immune systems [1–4]. Though human diseases
caused by this organism are substantial, its basic biology is
to date mostly obscure. In A. fumigatus, a large number of
proteins that play important roles in cell survival and
invasion are located in plasma membrane, endoplasmic
reticulum (ER), Golgi membrane, and other cytoplasmic
organelles. Glycosylation begins in the rough ER [5], as a
co- or post-translational event, and proceeds as the glyco-
proteins migrate through the Golgi to their final destination.
The elaboration of the carbohydrate groups depends on the
expression and subcellular localization of the specific
enzymes required for their biosynthesis and on the final
destination and rate of transport in individual glycoproteins
through the various compartments. Enzymes responsible
for cell wall organization and cell morphogenesis, such
as chitin synthase, b(1,3)-glucan synthase, a(1,3)-glucan
synthase, b(1,3)-glucanosyltransferases, mannosyltransfe-
rases, and chitinase are integral membrane proteins [6, 7].
Sphingosine-1-phosphate (S1P) phosphohydrolase 1 (SPP-1)
regulating sphingolipid metabolism and apoptosis is loca-
ted mainly in the endoplasmic reticulum [8].
Since such a large number of proteins associated with
glycoconjugates and cell wall biosynthesis are located in
cytoplasmic organelles as well as plasma membrane,
investigation of the structure and function of these proteins
in A. fumigatus proves to be increasingly significant. With
the recent accomplishment of genomic sequencing of
A. fumigatus [9], identification of membrane proteins on a
large scale becomes practical. To date, mass spectrometry
(MS)-based methods for the identification of proteins have
Electronic supplementary material The online version of thisarticle (doi:10.1007/s12033-009-9224-2) contains supplementarymaterial, which is available to authorized users.
H. Ouyang � Y. Luo (&) � L. Zhang � Y. Li � C. Jin (&)
State Key Laboratory of Microbial Resources, Institute
of Microbiology, Chinese Academy of Sciences,
A3 Datun Road, Chaoyang District, Beijing 100101, China
e-mail: luoym@im.ac.cn
C. Jin
e-mail: jinc@sun.im.ac.cn
Mol Biotechnol
DOI 10.1007/s12033-009-9224-2
been become a standard platform in proteomics. The most
popular MS-based strategies rely on proteolytic digestion
of proteins into peptides before introduction into the mass
spectrometer. Digestion of proteins into similar sized
peptides helps to overcome the solubility and handling
problems associated with ionization in the mass spec-
trometer. For complex mixtures of proteins which are not
sufficiently resolved in 1DE, a multidimensional separation
may be necessary in that components not separated in the
first dimension are separated in the second. The 2DE is the
most common multidimensional separation technique used
to separate complex mixtures. However, it has obvious
shortcomings such as limited dynamic range, inability to
detect membrane proteins and extremely basic or extre-
mely acidic proteins under standard conditions. Though
Kniemeyer et al. [10] used an optimized 2DE protocol to
improve the 2DE resolution and map quality of A. fumig-
atus, all of the proteins detected were basically cytoplasmic
proteins with low molecular weights. To increase the sol-
ubility of GPI-anchored membrane proteins in 2DE, Bru-
neau et al. [7] reported an applicable method to separate
GPI-anchored membrane proteins, in which the GPI-
anchored proteins were first released from membrane
preparation in a soluble form by an endogenous GPI-
phospholipase C and then separated by 2DE. Though
available for GPI-anchored proteins, this method still faces
challenges in separating the membrane proteins with
transmembrane segments and high MWs. To detect as
many membrane proteins potentially related to the syn-
thesis of glycoprotein, glycolipid, and cell wall in the
whole membrane system comprising plasma membrane
and cytoplasmic organelles, we used the currently devel-
oped multidimensional protein identification technology
(MudPIT) [11, 12] to directly analyze the total membrane
preparations collected by ultracentrifugation. In addition,
cell wall biosynthetic pathways have been recognized for a
long time as essential and unique specific drug targets and
the recent clinical launch of caspofungin, a b(1,3)-glucan
synthase inhibitor, has confirmed that the development of
new drugs can indeed be based on inhibition of cell-wall
biosynthetic enzymes [13]. In the light of the importance of
the biosynthetic pathways of cell wall and ergosterol in
antifungal drug design, we also briefly discussed the pro-
teins that may be used as drug targets.
Materials and Methods
Fungal Culture and Membrane Preparation
A. fumigatus, strain YJ407, was inoculated in complete
medium (0.1% yeast extract, 0.2% peptone, 1% glucose,
0.15% casein, and 2% of salt solution (v/v) containing 2.6%
KCl, 2.6% MgSO4 � 7H2O, 7.6% KH2PO4, 5% of trace-
element solution (v/v), pH 7.0) and grown at 37 �C for 24 h
in a 15-L fermentor (Bioflo410) with stirring (200 rpm) and
fresh air (1 L air min-1). A liter of trace-element solution
contains 40 mg NaB4O7 � 10H2O, 400 mg CuSO4 � 5H2O,
800 mg FeSO4 � 2H2O, 800 mg MnSO4 � 2H2O, 800 mg
Na2MO4 � 2H2O, and 8 g ZnSO4 � 7H2O.
The membrane preparation was performed according to
the method modified by Fontaine et al. [14]. Briefly, the
mycelium was collected by filtration under vacuum,
washed with water, and then ground in liquid nitrogen. The
power was resuspended in disruption buffer solution
(200 mmol/L Tris–HCl, 20 mmol/L EDTA, pH 8.0,
1 mmol/L phenylmethylsulfonyl fluoride buffer) at 4 �C in
presence of glass beads (0.5–0.75 mm diameter). Cell wall
was removed by centrifugation at 10,000g for 10 min at
4 �C. Total membrane pellets were then collected by
ultracentrifugation at 150,000g for 60 min at 4 �C. The
membrane pellets were resuspended in the disruption buf-
fer, homogenized, and then centrifuged at 150,000g for
60 min at 4 �C. The resuspension was repeated three times
to remove the contaminants from nonmembrane compo-
nents. The pellets were finally stored at -80 �C for future
use.
Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis and In-Gel Digestion
Two hundred micrograms of the total membrane prepara-
tion was dissolved in 29 loading buffer containing 4%
sodium dodecyl sulfate (SDS), 20% glycerol, 10%
2-mercaptoethanol, 0.004% Bromophenol Blue, and
0.125 mol/L Tris–HCl, pH 6.8, boiled for 5 min, and
subsequently run on a 12% SDS polyacrylamide gel
without sample loading comb. The gels were silver stained
as previously described [15]. The whole gel was cut for
each run and divided into 27 sections. When cutting the
gels, we have a strategy that the individual gel pieces with
similar staining strength or visually unstained were com-
bined together respectively, preventing interference of
identification of low abundance proteins from high abun-
dance proteins, as shown in Fig. 1. The stained gel pieces
were destained and submitted to in-gel digestion as previ-
ously described [15].
2D LC-MS/MS Analysis
The digested peptide mixtures for each sample were
desalted with Hypersep c18 SPE cartridge (Thermo Sci-
entific, Bellefonte, PA, USA). The cartridge was condi-
tioned before use by 5 9 1 mL of 0.1% TFA in 50%
acetonitrile and then by 5 9 1 mL of 0.1% TFA. The
peptide mixtures to be desalted were dissolved in 100 lL
Mol Biotechnol
of 0.1% TFA and passed through the conditioned cartridge.
The cartridge was washed three times with 1 mL of 0.1%
TFA and the peptides were eluted twice with 1 mL of 0.1%
TFA in 50% acetonitrile followed by evaporation to dry-
ness in a Speed-Vac. The desalted peptides were dissolved
in 20 lL of 0.1% formic acid and loaded on a strong cation
exchange column (BioBasic SCX, 0.32 mm 9 10 cm,
ThermoHypersil, Allentown, PA) via the autosampler, and
then step-eluted with a 10-step NH4Cl gradient (0, 10, 30,
50, 80, 100, 150, 180, 200, and 400 mM in 5% acetonitrile)
from the strong cation exchange column onto alternating
reversed phase columns (BioBasic C18, 300 A, 5 lm sil-
ica, 180 lm 9 10 cm, ThermoHypersil, Allentown, PA)
for the second dimensional separation. The flow rate for the
reversed phase separation was maintained at 100 lL/min
before splitting and at 1.0 lL/min after the flow split. The
gradient was started at 5% acetonitrile in 0.1% formic acid
for 20 min, then ramped to 50% acetonitrile within 80 min,
and then changed to 95% acetonitrile for 20 min, and
finally changed to 5% acetonitrile for an additional 20 min.
The resolved peptides were subjected to MS/MS analysis
with LCQ Deca XP Plus ion-trap mass spectrometer
(ThermoFinnigan, San Jose, CA) equipped with a nano-
spray source. The MS/MS data were acquired in the data-
dependent scan mode including four scan events: one full-
range MS scan and three MS/MS scans on the three most
intense precursors. The mass spectra were measured with
an overall mass/charge (m/z) range of 400–2000.
Database Searching
Each sample analyzed by 2D LC-MS/MS generates 10
peak list files, and 270 peak list files were created from 27
sections. All MS/MS spectra were searched using Thermo
Finnigan Bioworks 3.1 against the protein sequence data-
base of A. fumigatus downloaded from NCBI: http://www.
ncbi.nlm.nih.gov/ containing 21,395 protein entries, with a
static modification of ?57.0215 Da on cysteine residue and
a differential modification of 15.9994 on methionine. The
precursor ion mass tolerance was 1.4 Da, and the fragment
ion mass tolerance was 1.5 Da. We used the following
three steps to perform the data processing. First, we used
SEQUEST criteria below to perform an initial filtration:
DeltaCn C 0.1; Rsp C 1; Xcorr C 1.9 for singly charged
fragments; Xcorr C 2.2 for doubly charged; Xcorr C 3.75
for triply charged. Second, we used the AMASSv1.17.0.17
developed by Sun and co-workers [16, 17] (available at
http://www.proteomics-cams.com) to further filter the SE-
QUEST results based on three parameters: MatchPct C 60,
Cont C 40, and Rscore \ 2.6. Proteins with two or more
spectra approved by AMASS were accepted as positive
identifications. If a protein has multiple isoforms or has
multiple entries in the databases, we only specify the major
form of the protein unless a specific peptide points to a
region of the protein, which exists only in one of the
isoforms. Finally, reverse database searching was used
to estimate the false positive rate. The false positive
rate = peptide number in reverse database/peptide number
in forward database 9 100%.
Results
Proteins Located in Plasma Membrane
and Cytoplasmic Organelles
The results reported here are from merged data of 27
fractions, leading to the identification of 794 proteins from
the total membrane preparations of A. fumigatus. Reverse
database search revealed that the false positive rate
was 3.51% for positive peptides filtered by combined
SEQUEST/AMASS parameters. Of the 794 proteins, 530
were identified with at least two different peptides detected
with MS/MS spectra (Supplementary Table 1) and were
completely discussed. We assigned the subcellular loca-
tions of the 530 proteins according to the ‘‘GO_compo-
nent’’ in NCBI, and for those whose subcellular locations
are not labeled in NCBI, a WoLF PSORT software (freely
Fig. 1 SDS-PAGE of total membrane preparations of A. fumigatus.The left lane indicates the low Mr protein markers, and the right lane
indicates the 27 sections of A. fumigatus cut and divided based on
similar staining intensity
Mol Biotechnol
available at wolfpsort.org) was used to predict the sub-
cellular localization. Due to use of ultracentrifugation at
150,000g, the cytoplasmic organelles were collected as
well as plasma membrane pellets and therefore cytosolic
and excellular proteins located in cytoplasmic organelles
were identified along with the plasma membrane proteins.
The subcellular locations of the 530 proteins were pre-
dicted, and the subcelluar location distribution was shown
in Fig. 2.
As shown in Fig. 2, major the proteins are located in
ribosome, mitochondrion, nucleus, and plasma membrane,
accounting for 15.8, 30.4, 15.3, and 16.6%, respectively.
The others are located in the cytoplasmic organelles such
as endoplasmic reticulium (ER), golgi, vesicle, vacuole,
and proteasome complex. For the mitochondrial proteins
identified in this study, based on the annotation in NCBI,
27 are located in mitochondrial inner membrane, 7 in
mitochondrial outer membrane, 9 in mitochondrial matrix,
20 in mitochondrial ribosome, and 1 in mitochondrial inner
membrane space (Supplementary Table 2). It is necessary
to point out though 20 proteins identified in this study were
predicted as cytosolic proteins and 3 as excellular ones
by using the WoLF PSORT software (Supplementary
Table 1), these cytosolic and excellular proteins are not
caused by the contamination of nonmembrane components
for the total membrane pellets were strictly prepared
and washed three times with the disruption buffer (see
‘‘Materials and Methods’’ section). After three washes, the
supernatant was determined by SDS-PAGE to evaluate
washing efficiency and no obvious protein bands were
observed on the silver-stained gel, showing that the total
membrane isolation was reasonably successful. Actually, a
large number of cytosolic and excellular proteins were
identified due to their binding to membrane or transiently
bound to membrane, and their subcellular locations are
much dependent on their different functions, and therefore
multiple localization of the same protein is actually
reasonable [18]. As listed in Supplementary Table 3, 12
cytosolic proteins and 2 excellular proteins have been
reported to be associated with cytoplasmic organelles.
These extracellular and cytosolic proteins were collected
by ultracentrifugation in forms of cytoplasmic organelles
such as ER/golgi transport vesicles [19], sorting vacuoles
[20], degradation vacuoles [21], and storage vacuoles [22].
For example, Hsp70 chaperone (HscA) (gi|70983346) was
predicted as cytosolic protein in this study and was
reported to be associated with peroxisomes and involved in
import of proteins into organelles [23]. Glyceraldehyde
3-phosphate dehydrogenase (gi|70985278), as a typical
cytosolic protein identified in this study, is involved in the
vesicle transport from the ER to golgi [24].
One of the obvious advantages of 2D LC-MS/MS over
2DE is reflected in the identification of proteins with
extreme pIs and high MWs. Of the 530 identified proteins,
29 are with pIs \ 5 and 36 with pIs [ 11, accounting
for 5.5% and 6.8%, respectively, and 45 proteins are
with MWs [ 100 kD (Supplementary Table 1), accounting
for 8.5%, the largest being alpha-1,3-glucan synthase
(gi|70985813) with a MW of 274.4 kD, from which three
unique peptides were identified. However in the previous
0 5 10 15 20 25 30 35
ribosome
cytosol
mitochondrion
nucleus
vesicle
proteasome
extracelluar
golgi
ER
vacuole
plasma membrane
cytoskeleton complex
peroxisome
spliceosome
lipid particle
translation initiation factor complex
phenylalanine-tRNA ligase complex
microsome
Sub
cellu
lar
loca
tion
Percent (%)
Fig. 2 Percentage of proteins
located in plasma membrane
and cytoplasmic organelles
Mol Biotechnol
study, the proteome of A. fumigatus separated by 2DE were
major the cytoplasmic proteins with pIs 5.27–9.7 and
MWs \ 90 kD [10].
In this study, it is the proteins with high MWs that play
great roles in the synthesis of glycoconjugates and cell
wall. For example, five proteins with MWs [ 100 kD,
including chitin synthase G (gi|146322408, 102.4 kD),
chitin synthase E (gi|71001992, 206.6KD), a-1,3-glucan
synthase (gi|70985813, 274.4 kD), b-1,3-beta-glucan syn-
thase catalytic subunit FksP (gi|70992539, 219.8 kD), and
chitin synthase A (gi|70988943, 107.4 kD) are involved in
cell wall organization and biosynthesis. These proteins,
except chitin synthase A, are GPI-anchored membrane
proteins. Whereas in the previous report by Bruneau
et al. [7], the MWs of the identified GPI proteins are
below 80 kD. In addition, another two proteins with high
MWs, including GPI-anchor biosynthetic protein (Mcd4)
(gi|70982111, 114.4 kD) and protein mannosyltransferase 1
(gi|71000555, 107.1 kD), are identified to participate in
GPI anchor biosynthesis and O-glycosylation, respectively.
The above results suggest that 2D LC-MS/MS combined
with 1D SDS-PAGE is a powerful tool for identifying the
membrane proteins with extreme pIs and high MWs.
Membrane Proteins Involved in Glycosylation
of Proteins
So far, protein glycosylation in A. fumigatus is less
extensively studied. What we described here about the
functions and subcellular locations were much dependent
on Go_function and Go_component in NCBI and related
reports in yeast and other eukaryotic organisms [25]. As
summarized in Table 1, eleven ER- and four Golgi-bound
membrane proteins and two plasma membrane proteins
were identified to be involved in protein glycosylation
(N-glycosylation, O-glycosylation and GPI-anchoring). A
Dense Alignment Surface (DAS) method (available at
http://www.sbc.su.se/*miklos/DAS/) was used to predict
the transmembrane segments of these membrane proteins.
As shown in Table 1, when the cutoff for DAS score is set
at 2.2, at least one transmembrane segment is found in most
of membrane proteins except dolichol-phosphate manno-
syltransferase (gi|70999724) with no transmembrane
segment detected. It is crucial to point out the cDNA
sequences of four proteins have been cloned by our group,
leading to updated accession numbers (Table 1). Based on
the membrane proteins identified in this study, we were
able to develop glycosylation pathways in A. fumigatus
including N-glycosylation, O-mannosylation, and GPI
biosynthesis (Fig. 3).
N-glycosylation is initiated with the transfer of the
N-glycan precursor to nascent polypeptide, which is cata-
lyzed by oligosaccharyltransterase (OST). Biochemical and
genetic investigations have led to the discovery of nine
protein subunits of the OST in yeast, five of which are
essential for growth. In mammals, seven proteins have
meanwhile been identified as components of the OST.
Although the specific role of the individual subunits is not
well understood, some evidences strongly support the idea
that Stt3 is the catalytic subunit of OST [26]. In our study,
five subunits of the OST were identified and Stt3
(gi|70983446) was a putative catalytic subunit (Table 1 and
Fig. 3). Detailed descriptions about the structures, func-
tions, and localization of the OST in yeast and other
eukaryotic organisms can be seen everywhere [26–28].
Although we did not identify the proteins involved
in N-glycan trimming and processing, the calnexin
(gi|70993400), a protein that is involved in the correct
folding of secretory glycoproteins of mammalian cell [26],
was identified in our study, suggesting the existence of
N-glycan-dependent quality control system of protein
folding in A. fumigatus. Once the N-linked core oligosac-
charide is transferred to the Golgi, it is extended by the
addition of the outer chain. In yeast several proteins have
been shown to participate in the synthesis of this structure,
including Och1p, Mannan polymerase I complex (M-Pol I),
M-Pol II, Mnn2p, Mnn5p, Mnn4p, Mnn6p, and Mnn1p
[29]. M-Pol I and M-Pol II are a(1,6)-mannosyltransferases
and responsible for the elongation of the a(1,6)-linked
backbone of the outer chain. M-Pol I consists of Mnn9p
and Van1p, while M-Pol II is a multiprotein complex
including Mnn8p (Anp1p), Mnn9p, Mnn10p, Mnn11p, and
Hoc1p. In the membrane preparation of A. fumigatus, the
Kre5 (gi|70997373) and Ktr4 (gi|70985124) were identified
as a1,2-mannosyltransferases. However, only one subunit
of M-Pol I and M-Pol II, the Mnn9 (gi|70988857) was
discovered, which is consistent with the shorter N-glycan
observed in this species.
The structure of the O-glycans in peptidogalactomannan
of the cell wall of A. fumigatus has been determined
as Glca1-6Man1-O, Galfb1-6Mana1-6Man1-O, Galfb1-
5Galfb1-6 Mana1-6Man1-O, and Galfb1-5(Galfb1-5)3
Galfb1-6Man-O [30]. The protein O-mannosylation of
fungi is initiated in the ER by the transfer of mannose from
dolichyl-phosphate mannose. This reaction is catalyzed by
a family of protein O-mannosyltransferase (PMTs) [31].
Three putative PMTs (gi|70991332, gi|71000555, and
gi|70983460) were found in the membrane preparation of
A. fumigatus. In addition, two Golgi-located proteins, Kre2
(gi|70997663) and Ktr4 (gi|70985124), were identified as
a1,2-mannosyltransferases in O-glycosylation pathway. In
S. cerevisiae, KRE2 and KTR4 belong to the KRE2/MNT1
mannosyltransferase gene family. All of them are predicted
to be membrane proteins located in Golgi. The first
member of this family (KRE2/MNT1) is identified to
encode for an a1,2-mannosyltransferase involved in
Mol Biotechnol
Ta
ble
1M
emb
ran
ep
rote
ins
inv
olv
edin
pro
tein
gly
cosy
lati
on
Pro
tein
IDP
rote
inn
ame
Pep
tid
eC
har
ge
Xco
rrT
ran
smem
bra
ne
seg
men
taF
un
ctio
nS
ub
cell
ula
r
loca
liza
tio
n
Nu
cleo
tid
e
ID ann
ota
ted
by
ou
rg
rou
pb
gi|7
09
94
22
4O
lig
osa
cch
ary
ltra
nsf
eras
e
sub
un
itri
bo
ph
ori
nII
DL
PV
QF
LS
VA
EP
LD
AR
23
.26
75
4N
ER
TG
LD
ISY
PF
TV
K2
2.8
04
9
LP
EIH
HIF
K2
2.7
41
3
AH
QV
FL
LL
R2
2.6
43
5
VIL
TA
QE
GS
SA
K2
3.1
84
7
YG
KL
PE
IHH
IFK
22
.55
01
LV
IGS
FG
SS
HA
YN
EP
AF
PL
IVA
R3
4.1
95
6
AH
QV
FL
LL
RD
PK
23
.06
2
IPD
NK
PL
SK
PV
AL
GS
SD
TL
K3
3.9
16
8
NP
DE
PV
PT
VE
VS
R2
2.3
38
8
gi|7
09
98
01
6O
lig
osa
cch
ary
ltr
ansf
eras
e
sub
un
it(b
eta)
AV
PH
TL
GD
AN
PL
IAP
ILR
23
.40
97
3N
ER
RP
FL
TN
IEE
K2
2.8
78
7
VG
KIE
HH
LA
ED
GE
ITP
EK
LN
PK
35
.60
73
HF
AH
NE
YP
R2
2.4
29
LL
VV
LE
DA
TE
KE
LY
SK
24
.56
51
IEH
HL
AE
DG
EIT
PE
KL
NP
K3
4.5
34
7
DG
NV
LL
AL
SG
K2
3.5
04
1
IEH
HL
AE
DG
EIT
PE
K2
3.3
85
5
AF
FD
GE
GV
VA
FP
R2
3.8
24
3
HD
VL
LL
HR
PG
K2
3.5
86
3
GY
NL
DF
ES
PK
23
.18
14
GY
GP
SL
TP
K2
2.2
06
3
LN
LE
PV
R2
2.2
68
2
VG
KIE
HH
LA
ED
GE
ITP
EK
LN
PK
23
.72
63
AV
PH
TL
GD
AN
PL
IAP
ILR
33
.99
71
VG
KIE
HH
LA
ED
GE
ITP
EK
34
.51
06
gi|7
09
89
81
5O
lig
osa
cch
ary
ltr
ansf
eras
e
sub
un
it(g
amm
a)
SV
SP
VA
PIG
LT
DV
TY
HD
LT
SK
PR
23
.90
95
6N
ER
DF
HV
AV
LL
TA
TD
AR
23
.02
45
LL
LA
TL
DF
SN
GK
ET
FQ
K2
3.8
20
1
Mol Biotechnol
Ta
ble
1co
nti
nu
ed
Pro
tein
IDP
rote
inn
ame
Pep
tid
eC
har
ge
Xco
rrT
ran
smem
bra
ne
seg
men
taF
un
ctio
nS
ub
cell
ula
r
loca
liza
tio
n
Nu
cleo
tid
eID
ann
ota
ted
by
ou
rg
rou
pb
gi|7
09
89
71
7O
lig
osa
cch
ary
ltr
ansf
eras
e
sub
un
it(a
lph
a)
YE
FT
KP
VIT
AS
LL
ER
23
.11
81
3N
ER
VA
LP
AD
FK
PQ
QV
FK
22
.80
72
DL
EV
SH
WG
GN
LA
TE
ER
22
.93
85
VP
FIE
GF
KV
PE
GA
QY
EK
23
.55
55
NV
NL
VR
11
.93
28
KA
AT
GA
DS
YV
LK
22
.82
55
NV
QY
QV
LE
GA
SS
NG
LP
SS
SQ
IQS
HIS
K3
4.2
09
1
YR
PG
QP
PK
R2
2.2
15
8
YE
FT
KP
VIT
AS
LL
ER
34
.39
72
YR
PG
QP
PK
22
.20
68
gi|7
09
83
44
6O
lig
osa
cch
ary
ltr
ansf
eras
e
sub
un
it(S
tt3
)
DH
NQ
AV
AF
DK
22
.66
88
14
NE
R
VK
DL
DN
LG
R2
2.5
12
8
gi|7
09
97
37
3A
lph
a-1
,2-m
ann
osy
ltra
nsf
eras
e
(Kre
5),
pu
tati
ve
KN
NL
KS
K2
2.3
39
21
NG
olg
i
NK
EL
EG
VV
QS
LK
23
.65
99
RL
PS
TP
NL
PF
DD
SS
RE
K2
2.3
79
4
gi|7
09
85
12
4A
lph
a-1
,
2-m
ann
osy
ltra
nsf
eras
e(K
tr4
)
CT
PG
QF
YA
GA
PF
LA
K2
2.6
93
91
NG
olg
i
NE
EL
ED
LIS
TM
KD
LE
R2
3.5
07
6
gi|7
09
88
85
7A
lph
a-1
,6m
ann
osy
ltra
nsf
eras
e
sub
un
it(M
nn
9)
EG
NA
AL
AA
LE
AA
IAK
23
.57
03
2N
Go
lgi
NK
EG
NA
AL
AA
LE
AA
IAK
23
.61
62
gi|7
09
91
33
2*
Pro
tein
O-m
ann
osy
l
tran
sfer
ase
(PM
T2
)
NL
HS
HA
IPA
PIT
K2
3.2
85
81
2O
ER
gi|4
84
79
76
9
GT
EV
GK
DS
PL
EIA
VG
SR
23
.64
14
EF
YF
DV
HP
PL
GK
22
.71
84
gi|7
10
00
55
5*
Pro
tein
man
no
sylt
ran
sfer
ase
1(P
MT
1)
SK
ET
LQ
VK
PL
PR
23
.21
02
12
OE
Rg
i|4
84
79
75
3
HL
NT
QG
GY
LH
SH
AH
MY
PT
GS
K3
4.3
42
5
LY
HA
MT
HR
22
.30
1
HL
NT
QG
GY
LH
SH
AH
MY
PT
GS
K3
4.2
02
3
ET
LQ
VK
PL
PR
22
.39
5
FF
MD
VH
PP
LA
K2
3.0
47
5
gi|7
09
83
46
0*
Man
no
sylt
ran
sfer
ase
(PM
T4
)A
HG
SA
GH
FV
TA
K2
2.8
46
61
2O
ER
gi|1
10
82
58
28
SK
AH
GS
AG
HF
VT
AK
22
.49
77
gi|7
09
99
72
4D
oli
cho
l-p
ho
sph
ate
man
no
sylt
ran
sfer
ase
LG
GH
EIV
EY
LK
22
.50
84
0N
_O
ER
GN
YV
IIM
DA
DF
SH
HP
K2
3.8
12
6
VA
EC
PIT
FV
DR
22
.58
57
QL
QT
LW
GS
EH
INL
KP
R2
2.4
53
Mol Biotechnol
synthesis of O-linked oligosaccharide. Further functional
investigation revealed that KRE2 participates in synthesis
of both O- and N-linked oligosaccharides. Kre2p/Mnt1p,
Ktr1p, and Ktr3p, have overlapping roles, and collectively
add most of the second and the third a(1, 2)-linked mannose
residues on O-linked oligosaccharides as well as some of the
a(1, 2)-linked mannoses in the branches of N-linked oligo-
saccharides [32]. Based on the O-glycan structures identi-
fied in A. fumigatus, the Kre2 (gi|70997663) and Ktr4
(gi|70985124) might be involved in the addition of the
second a(1,6)-linked mannose residue, instead of a(1,2)-
linked mannose residue in yeast. No b-Galf transferase was
identified in this study. Also, a genome analysis has failed to
find eukaryotic gene homologous to b-Galf transferase [31].
Thus, it is likely that A. fumigatus has a novel b-Galf
transferase.
Like most other eukaryotes, A. fumigatus harbors a
GPI-anchoring machinery and uses it to attach proteins to
membranes. While a few GPI proteins reside permanently
at the plasma membrane, a majority of them gets further
processed and is integrated into the cell wall by a covalent
attachment to cell wall glucans. The GPI biosynthetic
pathway is necessary for growth and morphogenesis [33].
In S. cerevisiae, the GPI lipids are synthesized in the ER
and added onto proteins by a pathway comprising 12 steps,
carried out by 23 gene products, 19 of which are essential.
Some of the estimated 60 GPI proteins predicted from the
genome sequence serve enzymatic functions required for
the biosynthesis and the continuous shape adaptations of
the cell wall, others seem to be structural elements of
the cell wall and yet others mediate cell adhesion [34]. It
has been shown that A. fumigatus GPI glycan moiety is
mainly a linear pentomannose structure linked to a glu-
cosamine residue: Mana1-3Mana1-2 Mana1-2Mana1-
6Mana1-4GlcN [14]. However, in contrast to yeast, little
is known for the GPI biosynthesis in A. fumigatus. Several
proteins that might be involved in the GPI biosynthesis
were identified in this study. A protein (gi|14632406) was
identified as a putative mannosyltransferase Smp3, an
enzyme that catalyzes the addition of the Man4 and is
essential in yeast and in C. albicans. Two GPI trans-
amidases, Gpi16p and PIG-S/Gpi17p, may catalyze the
transamidation reaction that results in the cleavage of the
polypeptide chain and the concomitant transfer of the GPI
anchor to the newly formed carboxy-terminal amino acid
of the anchored protein [35]. In addition, a plasma mem-
brane-bound protein, GPI-anchor biosynthetic protein
(Mcd4), (gi|70982111) was identified to be involved in
GPI anchor biosynthesis. It is well known that glycocon-
jugate biosynthesis requires activated form of monosac-
charide as sugar donor. In this study, we also identified
membrane proteins that were involved in specific steps of
galactose metabolism, glycosis, and mannose metabolismTa
ble
1co
nti
nu
ed
Pro
tein
IDP
rote
inn
ame
Pep
tid
eC
har
ge
Xco
rrT
ran
smem
bra
ne
seg
men
taF
un
ctio
nS
ub
cell
ula
r
loca
liza
tio
n
Nu
cleo
tid
eID
ann
ota
ted
by
ou
rg
rou
pb
gi|1
46
32
40
61
Man
no
sylt
ran
sfer
ase
(Sm
p3
)S
PII
TP
LN
NL
LY
NT
K2
2.2
45
51
0G
PI
ER
VS
AT
VL
WW
K2
2.3
65
8
gi|7
09
91
88
3G
PI
tran
sam
idas
eco
mp
on
ent
Gp
i16
AL
GQ
ILQ
HT
HT
K2
2.7
44
43
GP
IE
R
LS
SA
SG
NT
IAH
DT
PIY
MR
23
.98
65
gi|7
09
82
11
1*
GP
I-an
cho
rb
iosy
nth
etic
pro
tein
(Mcd
4)
VL
SH
GT
FG
VS
HT
R2
2.6
24
21
6G
PI
Pla
sma
mem
bra
ne
gi|9
00
18
75
4
AL
AA
LA
NT
QE
VL
EM
YR
22
.37
57
gi|7
09
91
52
5G
PI
tran
sam
idas
eco
mp
on
ent
PIG
-
S/G
Pi1
7
TT
QH
TL
DD
LN
EF
SA
HH
LR
34
.16
47
4G
PI
Pla
sma
VA
VY
LP
LL
GP
IGV
PL
VV
GL
LK
34
.05
48
Mem
bra
ne
gi|7
09
97
66
3al
ph
a-1
,2-m
ann
osy
ltra
nsf
eras
e
(Kre
2)
AH
ND
AP
PK
PP
VK
22
.45
36
1N
_O
Go
lgi
CH
CN
PS
EN
FD
WK
22
.58
47
NN
-gly
cosy
lati
on
,O
O-g
lyco
syla
tio
n,
N_
Ob
oth
N-g
lyco
syla
tio
nan
dO
-gly
cosy
lati
on
,E
Ren
do
pla
smic
reti
culu
m,
GP
IG
PI
anch
or
bio
syn
thes
isa
AD
ense
Ali
gn
men
tS
urf
ace
(DA
S)
met
ho
d(h
ttp
://w
ww
.sb
c.su
.se/*
mik
los/
DA
S/)
isu
sed
top
red
ict
tran
smem
bra
ne
alp
ha-
hel
ices
bas
edo
nsi
ng
lese
qu
ence
info
rmat
ion
,th
ecu
toff
for
DA
S
sco
reis
set
at2
.2b
Th
ecD
NA
seq
uen
ceo
fth
ep
rote
inis
clo
ned
fro
mA
.fu
mig
atu
sst
rain
YJ4
07
by
ou
rg
rou
p,
lead
ing
tou
pd
ated
acce
ssio
nn
um
ber
Mol Biotechnol
providing the sugar monomers, such as glucose, galactose,
mannose, and fructose for O-mannosylation, N-glycosyla-
tion, and GPI biosynthesis (Fig. 3). The activated forms of
sugar donors are indicated with gray background.
Membrane Proteins Involved in Sphingolipid Metabolic
Pathway
Glycosphingolipids (GSL) are essential components of the
fungal cell membrane. The sphingolipid moieties of GSL
contains a hydrophobic segment (ceramide), which is a
long-chain base (LCB) that is N-acylated with a very long-
chain a-hydroxy fatty acid, linked to various polar head
groups. It has been shown that A. fumigatus expresses both
glucosylceramide and galactosylceramide (GlcCer and
GalCer). The synthesis of GlcCer is essential for normal
development of A. fumigatus [36]. A comparative genome
analysis of the sphingolipid biosynthetic pathway in fungal
species shows that A. fumigatus has most of the sphingolipid
pathway genes found in other fungi, except for the CSG2
and IPT1 genes; the former is involved in the mannosylation
of inositol phosphorylceramide (IPC) to mannose–inositol–
phosphorylceramide and the latter involved in the synthesis
of mannose–(inositol-P)2–ceramide from mannose–inosi-
tol–phosphorylceramide [37]. Seven proteins involved in
specific steps of sphingolipid metabolic pathway are
detailed in Fig. 4 and the peptide fragments, charges, and
scores that are used to identify these proteins are listed in
Supplementary Table 4. Sphingolipid synthesis begins with
the condensation of palmitol-CoA and serine to yield
3-ketodihydrosphingosine, which is reduced to yield
dihydrosphingosine (DHS). The condensation reaction
is catalyzed by serine palmitoyltransferases, LcbAp
(gi|70992537), and Lcb2p (gi|70995600). Then 3-keto-
dihydrosphingosine is reduced by a 3-ketosphingosine
reductase, Tsc10p (gi|146322596) to produce the LCB. The
next step in sphingolipid synthesis, DHS is converted to
PHS. Sur2p (gi|70996596) is required for the hydroxylation
of DHS at C-4. Two fatty acid hydroxylases (gi|70999962
and gi|70996386) may be involved in the hydroxylation of
the very long-chain fatty acid. In addition, one putative S1P
lyase (gi|70993864) was also identified.
Fig. 3 Metabolic pathways of N-glycosylation, O-glycosylation, and
GPI biosynthesis in A. fumigatus. Glycolysis, mannose metabolism,
and galactose metabolism provide sugar monomers including
GlcNAc, Man, galactose, fructose, and Glu for the biosynthesis of
N-glycans, O-glycans, and GPI. The proteins involved are detailed in
specific steps of individual metabolic pathways. The activated donors
of monosaccharides are indicated with gray background. Dolichyl-
phosphate-mannose, which is generated from mannose metabolism, is
used as donor of mannoses in GPI biosynthesis and O-mannosylation
Mol Biotechnol
Membrane Proteins Involved in Cell Wall Biosynthesis
We successfully found nine plasma membrane-bound
proteins involved in cell wall biosynthesis (Fig. 5 and
Supplementary Table 5), four of which were identified as
chitin synthases (CHSs), including CHSA (gi|70988943),
CHSC (gi|70985514), CHSE (gi|71001992), and CHSG
(gi|70998925), suggesting additive, redundant, or syner-
gistic roles of the various CHSs of this multigene family in
chitin deposition. Indeed, it has been shown that the
mutations in CHSE and CHSG result in a growth pheno-
type such as reduction in hyphal growth, periodic swellings
along the length of hyphae and a block on conidiation
partially restored by growth in presence of an osmotic
stabilizer. While a double CHSE/CHSG disruption mutant
is still viable, suggesting an alternate regulation of the
various remaining CHSs [6].
The Fksp (gi|70992539) was identified as a b-1,3-glucan
synthase. This protein might be essential since only one
FKS gene has been found in A. fumigatus. A protein
gi|70985813 was identified as a a-1,3-glucan synthase
responsible for a(1,3)-glucan synthesis. This enzyme
internally splits a b(1,3)-glucan molecule and transfers the
newly generated reducing end to the nonreducing end of
another b(1,3)-glucan molecule. The Gel1p (gi|70988799)
was identified as a GPI-anchored b1,3-glucanosyltransfer-
ase that generates a new b(1,3)-linkage to allow the elon-
gation of b(1,3)-glucan chains [38, 39]. Besides, the Mnn9
(gi|70988857) may be involved in the biosynthesis of
galactomannan of cell wall, as the yeast Mnn9p has been
shown to be responsible for the elongation of the mannan
chain and involved in the septum formation [40].
When talking about the proteins involved in cell wall
biosynthesis, we will have to mention their roles in
Fig. 4 Diagram of sphingolipid
metabolism in A. fumigatus. In
our study, seven proteins with
accession numbers were
identified to be involved in five
sequential reaction steps of
sphingolipid synthesis, which
begins with the condensation of
palmitoyl-CoA and serine
D-MannoseD-Gluocosamine-6Pα−Glucoseβ−GlucoseD-Galactose
Fig. 5 Diagram of cell wall
biosynthetic pathway. D-
Glucosamine-6P generated in
glycosis is transformed to UDP-
GlcNAc in aminosugars
metabolism and used as sugar
donors for chitin biosynthesis.
Glycosis and galactose
metabolism provide Gal and
Man for galactomannan
biosynthesis, a-glucose for
a-glucans biosynthesis, and
b-glucose for b-glucan
biosynthesis
Mol Biotechnol
antifungal drug design. As fungal cell wall polysaccharides
do not have a counterpart in humans, theoretically, those
proteins involved in cell wall biosynthesis can be used as
ideal antifungal drug targets, as in the case of caspofungin,
a new antifungal that is designed as a b(1,3)-glucan
synthase inhibitor [13]. As shown in Fig. 5, four chitin
synthases are involved in chitin biosynthesis, Mnn
(gi|70998857) is involved in galactomannan biosynthesis,
AGS (gi|70985813) is involved in a1,3 glucan biosynthesis,
and Gel1p (70988799) and Fksp (gi|70992539) are
involved in b-1,3 glucan biosynthesis, the inhibitors of
these enzymes that can disrupt the cell wall biosynthesis in
fungi may be potentially used as drug targets. And to some
extent, development of new antifungals based on inhibition
of cell wall biosynthesis may solve the current problem in
resistance to azole antifungals [41].
In addition, in this study, we successfully found six ER-
bound enzymes (Supplementary Table 6) that are associ-
ated with antifungal drug design based on inhibition of the
formation of cell membrane by blocking the ergosterol
biosynthetic pathway [41], which provides another alter-
native method to overcome the resistance to azole anti-
fungals. As shown in Fig. 6, two sterol c14-reductases
(gi|66847697, gi|66847953) were identified in this study
and have been successfully used as drug target of
amorolfine, an antifungal [42]. As in the case of azoles
directed against C14a-demethylase in the ergosterol path-
way, design of the inhibitors directed against the identi-
fied enzymes, farnesyl-diphosphate farnesyltransferase
(gi|66844434), Erg26 (gi|66853474), Erg28 (gi|66853125),
and Erg3 (gi|66845190), should be a reasonable starting
point for new antifungal development, which will effec-
tively promote the antifungal drug discovery based on
inhibition of ergosterol biosynthesis.
Besides the proteins described above, many other pro-
teins that are localized in ubiquitous cytoplasmic organ-
elles also fulfill extensive functions in A. fumigatus. Since
our current interest is focused on membrane proteins
related to the synthesis of glycoconjugates and cell wall,
the proteins that are putatively involved in cell adhesion,
Fig. 6 Ergosterol biosynthetic pathway. Six enzymes indicated with accession number are identified in this study. The enzymes involved in the
specific steps of ergosterol biosynthesis can potentially be used as drug targets for antifungal drug design
Mol Biotechnol
regulation of autophagy, cell cycling, SNARE interaction
in vesicular transport system, regulation of actin cyto-
skeleton, ubiquitin mediated proteolysis, phosphatidylino-
sitol signaling pathway, MAPK signaling pathway, ERBB
pathway, calcium signaling pathway, and other signaling
pathways are not discussed here.
Discussion
In A. fumigatus, a large number of membrane proteins that
fulfill different functions are located in ubiquitous cyto-
plasmic organelles as well as cell membrane. In order to
get a global view of the membrane proteins involved in
biosynthesis of cell wall and glycoconjugates, the total
membrane pellets were directly subjected to one-dimen-
sional SDS-PAGE followed by 2D LC-MS/MS capable of
high throughput separation and identification. Many of the
membrane proteins involved in protein glycosylation, cell
wall biosynthesis, and glycolipid synthesis were identified
to be localized in endoplasmic reticulum, Golgi apparatus,
and other cytoplasmic organelles as well as plasma mem-
brane, strongly demonstrating the availability and necessity
of this method. Bands from 1D gels were cut according to
invisibly distinguished abundance to achieve the enrich-
ment of low abundance proteins, avoiding the interference
of high abundance proteins. Besides, we combined 1DE
with 2D LC-MS/MS rather than directly subjected the total
membrane preparations to 2D LC-MS/MS. In this way, in-
gel digestion was carried out in 1D gel instead of in-
solution. The major problem in in-solution digestion is the
introduction of denature reagent (8 mol/L urea or 6 mol/L
guanidine hydrochloride) to dissolve the proteins, whereas
the denatured protein solution must be diluted to below
2 mol/L urea or 1 mol/L guanidine hydrochloride prior to
proteolytic digestion to maintain the activity of the trypsin,
leading to loss of proteins caused by dilution precipitation.
Identification of total membrane proteome of A. fumigatus
provides only a starting point for functional analysis. We will
hopefully continue to make progresses in deciphering the
structures and functions of membrane proteins related to
glycoconjugates in A. fumigatus as we have been doing [33].
Acknowledgments This project was supported by the State ‘‘863’’
High-tech Project (2007AA02Z164), the National Basic Research
Program of China (2006CB504400), and the Youth Fund from Institute
of Microbiology, Chinese Academy of Sciences (0654041005).
References
1. Denning, D. W., Anderson, M. J., Turner, G., Latge, J. P., &
Bennett, J. W. (2002). Sequencing the Aspergillus fumigatusgenome. The Lancet Infectious Diseases, 2(4), 251–253.
2. Casadevall, A., & Pirofski, L. A. (1999). Host-pathogen inter-
actions: Redefining the basic concepts of virulence and patho-
genicity. Infection and Immunity, 67(8), 3703–3713.
3. Denning, D. W. (1998). Invasive aspergillosis. Clinical InfectiousDiseases, 26(4), 781–803.
4. Greenberger, P. A. (2002). Allergic bronchopulmonary aspergil-
losis. Journal of Allergy and Clinical Immunology, 110(5), 685–
692.
5. Kukuruzinska, M. A., Bergh, M. L., & Jackson, B. J. (1987).
Protein glycosylation in yeast. Annual Review of Biochemistry,56, 915–944.
6. Bernard, M., & Latge, J. P. (2001). Aspergillus fumigatuscell wall: Composition and biosynthesis. Medical Mycology,39(Suppl 1), 9–17.
7. Bruneau, J. M., Magnin, T., Tagat, E., Legrand, R., Bernard, M.,
Diaquin, M., et al. (2001). Proteome analysis of Aspergillus fu-migatus identifies glycosylphosphatidylinositol-anchored proteins
associated to the cell wall biosynthesis. Electrophoresis, 22(13),
2812–2823.
8. Le, S. H., Galve-Roperh, I., Peterson, C., Milstien, S., & Spiegel,
S. (2002). Sphingosine-1-phosphate phosphohydrolase in regu-
lation of sphingolipid metabolism and apoptosis. Journal of CellBiology, 158(6), 1039–1049.
9. Nierman, W. C., Pain, A., Anderson, M. J., Wortman, J. R., &
Kim, H. S. (2005). Genomic sequence of the pathogenic and
allergenic filamentous fungus Aspergillus fumigatus. Nature,438(7071), 1151–1156.
10. Kniemeyer, O., Lessing, F., Scheibner, O., Hertweck, C., &
Brakhage, A. A. (2006). Optimisation of a 2-D gel electropho-
resis protocol for the human-pathogenic fungus Aspergillusfumigatus. Current Genetics, 49(3), 178–189.
11. Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., Mize, G. J.,
Morris, D. R., et al. (1999). Direct analysis of protein complexes
using mass spectrometry. Nature Biotechnology, 17(7), 676–682.
12. Washburn, M. P., Wolters, D., & Yates, R., Jr. (2001). Large-
scale analysis of the yeast proteome by multidimensional protein
identification technology. Nature Biotechnology, 19(3), 242–247.
13. Abruzzo, G. K., Gill, C. J., Flattery, A. M., Kong, L., Leighton,
C., Smith, J. G., et al. (2000). Efficacy of the echinocandin ca-
spofungin against disseminated aspergillosis and candidiasis in
cyclophosphamide-induced immunosuppressed mice. Antimicro-bial Agents and Chemotherapy, 44(9), 2310–2318.
14. Fontaine, T., Magnin, T., Melhert, A., Lamont, D., Latge, J. P.,
& Ferguson, M. A. (2003). Structures of the glycosylpho-
sphatidylinositol membrane anchors from Aspergillus fumigatusmembrane proteins. Glycobiology, 13(3), 169–177.
15. Gharahdaghi, F., Weinberg, C. R., Meagher, D. A., Imai, B. S., &
Mische, S. M. (1999). Mass spectrometric identification of pro-
teins from silver-stained polyacrylamide gel: A method for the
removal of silver ions to enhance sensitivity. Electrophoresis,20(3), 601–605.
16. Sun, W., Li, F., Wang, J., Zheng, D., & Gao, Y. (2004). AMASS:
Software for automatically validating the quality of MS/MS
spectrum from SEQUEST results. Molecular & Cellular Pro-teomics, 3(12), 1194–1199.
17. Li, F., Sun, W., Gao, Y., & Wang, J. (2004). RScore: A peptide
randomicity score for evaluating tandem mass spectra. RapidCommunications in Mass Spectrometry, 18(14), 1655–1659.
18. Sirover, M. A. (1999). New insights into an old protein: The
functional diversity of mammalian glyceraldehyde-3-phosphate
dehydrogenase. Biochimica et Biophysica Acta, 1432(2), 159–
184.
19. Sulli, C., Fang, Z., Muchhal, U., & Schwartzbach, S. D. (1999).
Topology of Euglena chloroplast protein precursors within
endoplasmic reticulum to Golgi to chloroplast transport vesicles.
Journal of Biological Chemistry, 274(1), 457–463.
Mol Biotechnol
20. Seeger, M., & Payne, G. S. (1992). A role for clathrin in the
sorting of vacuolar proteins in the Golgi complex of yeast. EMBOJournal, 11(8), 2811–2818.
21. Egner, R., Thumm, M., Straub, M., Simeon, A., Schuller, H. J., &
Wolf, D. H. (1993). Tracing intracellular proteolytic pathways.
Proteolysis of fatty acid synthase and other cytoplasmic proteins
in the yeast Saccharomyces cerevisiae. Journal of BiologicalChemistry, 268(36), 27269–27276.
22. Burre, J., & Volknandt, W. (2007). The synaptic vesicle prote-
ome. Journal of Neurochemistry, 101(6), 1448–1462.
23. Walton, P. A., Wendland, M., Subramani, S., Rachubinski, R. A.,
& Welch, W. J. (1994). Involvement of 70-kD heat-shock pro-
teins in peroxisomal import. Journal of Cell Biology, 125(5),
1037–1046.
24. Tisdale, E. J., Kelly, C., & Artalejo, C. R. (2004). Glycer-
aldehyde-3-phosphate dehydrogenase interacts with Rab2 and
plays an essential role in endoplasmic reticulum to Golgi trans-
port exclusive of its glycolytic activity. Journal of BiologicalChemistry, 279(52), 54046–54052.
25. Yan, A., & Lennarz, W. J. (2005). Unraveling the mechanism of
protein N-glycosylation. Journal of Biological Chemistry, 280(5),
3121–3124.
26. Lehle, L., Strahl, S., & Tanner, W. (2006). Protein glycosylation,
conserved from yeast to man: A model organism helps elucidate
congenital human diseases. Angewandte Chemie, 45(41), 6802–
6818. (International ed. in English).
27. Knauer, R., & Lehle, L. (1999). The oligosaccharyltransferase
complex from yeast. Biochimica et Biophysica Acta, 1426(2),
259–273.
28. Kelleher, D. J., & Gilmore, R. (2006). An evolving view of the
eukaryotic oligosaccharyltransferase. Glycobiology, 16(4), 47R–
62R.
29. Munro, S. (2001). What can yeast tell us about N-linked glyco-
sylation in the Golgi apparatus? FEBS Letters, 498(2–3), 223–
227.
30. Leitao, E. A., Bittencourt, V. C., Haido, R. M., Valente, A. P.,
Peter-Katalinic, J., Letzel, M., et al. (2003). Beta-galactofur-
anose-containing O-linked oligosaccharides present in the cell
wall peptidogalactomannan of Aspergillus fumigatus contain
immunodominant epitopes. Glycobiol, 13(10), 681–692.
31. Goto, M. (2007). Protein O-glycosylation in fungi: Diverse
structures and multiple functions. Bioscience, Biotechnology, andBiochemistry, 71(6), 1415–1427.
32. Lussier, M., Sdicu, A. M., & Bussey, H. (1999). The KTR and
MNN1 mannosyltransferase families of Saccharomyces cerevi-siae. Biochimica et Biophysica Acta, 1426(2), 323–334.
33. Li, H., Zhou, H., Luo, Y., Ouyang, H., Hu, H., & Jin, C. (2007).
Glycosylphosphatidylinositol (GPI) anchor is required in Asper-gillus fumigatus for morphogenesis and virulence. MolecularMicrobiology, 64(4), 1014–1027.
34. Pittet, M., & Conzelmann, A. (2007). Biosynthesis and function
of GPI proteins in the yeast Saccharomyces cerevisiae. Biochi-mica et Biophysica Acta, 1771(3), 405–420.
35. Maxwell, S. E., Ramalingam, S., Gerber, L. D., Brink, L., &
Udenfriend, S. (1995). An active carbonyl formed during gly-
cosylphosphatidylinositol addition to a protein is evidence of
catalysis by a transamidase. Journal of Biological Chemistry,270(33), 19576–19582.
36. Levery, S. B., Momany, M., Lindsey, R., Toledo, M. S., Shay-
man, J. A., Fuller, M., et al. (2002). Disruption of the glucosyl-
ceramide biosynthetic pathway in Aspergillus nidulans and
Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide glu-
cosyltransferase strongly affects spore germination, cell cycle,
and hyphal growth. FEBS Letters, 525(1–3), 59–64.
37. Do, J. H., Park, T. K., & Choi, D. K. (2005). A computational
approach to the inference of sphingolipid pathways from the gen-
ome of Aspergillus fumigatus. Current Genetics, 48(2), 134–141.
38. Mouyna, I., Fontaine, T., Vai, M., Monod, M., Fonzi, W. A., Di-
aquin, M., et al. (2000). Glycosylphosphatidylinositol-anchored
glucanosyltransferases play an active role in the biosynthesis of the
fungal cell wall. Journal of Biological Chemistry, 275(20), 14882–
14889.
39. Mouyna, I., Morelle, W., Vai, M., Monod, M., Lechenne, B.,
Fontaine, T., et al. (2005). Deletion of GEL2 encoding for a
beta(1–3)glucanosyltransferase affects morphogenesis and viru-
lence in Aspergillus fumigatus. Molecular Microbiology, 56(6),
1675–1688.
40. Schmidt, M., Strenk, M. E., Boyer, M. P., & Fritsch, B. J. (2005).
Importance of cell wall mannoproteins for septum formation in
Saccharomyces cerevisiae. Yeast, 22(9), 715–723.
41. Veen, M., & Lang, C. (2005). Interactions of the ergosterol
biosynthetic pathway with other lipid pathways. BiochemicalSociety Transactions, 33(Pt 5), 1178–1181.
42. Lupetti, A., Danesi, R., Campa, M., Del, T. M., & Kelly, S.
(2002). Molecular basis of resistance to azole antifungals. Trendsin Molecular Medicine, 8(2), 76–81.
Mol Biotechnol
top related