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INTRODUCTION |
The fungal cell wall, which is
specific and essential to fungal life, is mainly constituted of
polysaccharides. Among all polysaccharides identified to date in the
cell wall, 
(1-3) glucans are the most prevalent, and they are
present in all yeast and filamentous fungi investigated to date
(14). Although (1-3) glucan biosynthesis has been the
subject of intensive research efforts for the last 30 years, the
(1-3) glucan biosynthetic pathway is not fully understood. It has
been known since the early studies of Cabib and coworkers (22,
31, 35, 36) that (1-3) glucans are synthesized from UDP
glucose by a membrane protein complex, (1-3) glucan synthase (EC
2.4.1.34;
UDP-glucose/liter 1,3--D-glucan-3--D-glucosyltransferase).
Synthesis occurs on the cytoplasmic side of the plasma membrane,
and (1-3) glucan chains are extruded towards the periplasmic space
(15, 35). The glucan synthase complex has been
characterized at the molecular level almost exclusively in the yeast
Saccharomyces cerevisiae (5, 7, 12, 19, 29) and
has been shown to be composed of two proteins: (i) the putative
catalytic subunit Fksp, a large-molecular-size (>200 kDa) polypeptide
with 16 transmembrane domains (12, 29, 30), and (ii) the
regulatory subunit Rho1p, a small-molecular-size GTPase, which
stimulates (1-3) glucan synthase activity in its prenylated form
(1, 11, 17, 18, 24, 28, 33).
If the (1-3) glucan synthase has been extensively analyzed in
yeast, then this enzymatic complex has been poorly studied in
filamentous fungi. Only one FKS gene had been cloned and
sequenced to date in Aspergillus nidulans (23),
and neither has a regulatory partner been identified nor has the
cellular localization of the glucan synthase complex been investigated.
This study was centered on the characterization of the glucan synthase
complex of the filamentous fungus Aspergillus fumigatus, which is a human opportunistic pathogen of increasing importance in
human health (26). Here we report (i) the cloning and
sequencing of the FKS1 and RHO genes, (ii) the
identification of the major proteins which coprecipitate with the
Fks1p-Rho1p-(1-3) glucan complex during product entrapment
experiments, and (iii) the localization of the glucan synthase complex
at the apices of hyphae.
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MATERIALS AND METHODS |
Strains and culture media.
Strains CBS 143.89 and 2965B2
were A. fumigatus clinical isolates. The strains were
maintained on 2% malt agar slants. Mycelia for DNA extraction were
grown for 18 h at 37°C in Sabouraud medium (2% glucose, 1%
mycopeptone) (Biokar). Mycelia for glucan synthase assays were produced
in the same medium in 2 liters of Biolafitte fermenter at 25°C for 16 h with an agitation of 500 rpm and an aeration of 0.5 liters of air/min
(2). Escherichia coli strain DH5
(Biolabs)
was used for cloning procedures with pBluescript SK(+) plasmid
(Stratagene), and E. coli strain BL21 (Pharmacia) was used
for expression with the pGEX4T vector (Pharmacia). Pichia pastoris strain SMD1168 (Invitrogen) was used for expression with the pPIC3 vector (Invitrogen).
Cloning procedures for A. fumigatus FKS1.
Approximately 50,000 recombinant plaques of an A. fumigatus
genomic library in 
EMBL3 (Stratagene) (a gift of M. Monod, CHUV, Lausanne, Switzerland) were immobilized on nylon membranes (Genescreen; Dupont NEN). These filters were probed with a
[-32P]dCTP-labeled 3.5-kb
(KpnI-KpnI) fragment of S. cerevisiae
FKS1, provided by A. F. J. Ram (Institute for Molecular
Cell Biology, University of Amsterdam, Amsterdam, The Netherlands),
under low-stringency hybridization conditions (hybridization and
washings at 50°C) (32). Positive plaques were purified,
and the DNA was isolated. Agarose gel electrophoresis of restricted
recombinant bacteriophage, Southern blotting, and cloning of the
positive bands in pBluescript SK(+) plasmid were performed according to
standard protocols (34). cDNA of FKS1 was
obtained by PCR using a gt11 (Stratagene) A. fumigatus
cDNA library (a kind gift of M. Monod) as template.
Cloning procedure for A. fumigatus RHO.
To clone
RHO genes, degenerated oligonucleotide primers
5'-GG(TC)GA(TC)GG(TC)GC(TC)TG(TC)GG(TC)AA-3' and
5'-TC(TC)TC(TC)TGGCCGGC(I)GT(GA)TCCCA(I)AG-3' were designed
based on conserved GTP binding and GTP hydrolysis sequences. Primers
were used in PCR with the genomic DNA phage library of A. fumigatus as template. An amplified DNA fragment from A. fumigatus genomic DNA was cloned, sequenced, and subsequently used
to screen the genomic library. cDNA of RHO genes were
obtained by PCR using the gt11 A. fumigatus cDNA library.
Sequencing and sequence analysis of A. fumigatus FKS1
and RHO genes.
Sequencing of FKS1 and
RHO1 from genomic DNA and cDNA was performed at ESGS
(Cybergène, Evry, France). DNA sequence data were analyzed using
the University of Wisconsin Genetics Computer Group programs
(10).
Southern blottings were performed to look for the presence of homologs
of AfFKS1 in the A. fumigatus genome. A. fumigatus genomic DNA was digested with BamHI,
ClaI, HindIII, and SalI, and the
blot was probed with a HindIII fragment of the
AfFKS1 gene (bases 1257 to 2354 from the genomic sequence)
under low-stringency hybridization conditions (hybridization and
washings at 42°C) (32).
Expression of AfFKS1.
The expression of the
conserved hydrophilic internal region (IntF) of AfFKS1 was
undertaken in E. coli strain BL21 using the expression
plasmid pGEX4T1. The IntF fragment (nucleotides [nt] 2943 to 4219)
was obtained by PCR using the primers Intgex1,
EcoRI
5'-AA
CAAGACTGAGTTCTTCCC-3'
(nt 2943 to 2962), and Intgex2,
NotI
5'-AT
CACGAATCATGGCAGGCA-3'
(antisense, nt 4201 to 4219), and the cDNA of
AfFKS1. The PCR fragment was then digested by
EcoRI-NotI. The IntF fragment was cloned into the
EcoRI/NotI site of pGEX-4T1 in fusion with glutathione S-transferase (GST). After expression following
the manufacturer's instructions (Pharmacia), E. coli
producing IntF-GST was resuspended in STE buffer (10 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA) supplemented with 1 mg of lysozyme (Sigma) per ml.
After 15 min at 0°C, the extract was sonicated for 1 min in the
presence of 1.5% (vol/vol) Sarkosyl to separate the recombinant
peptide from the inclusion bodies. After centrifugation at
13,000 × g, 2% (vol/vol) Triton X-100 was added to
the supernatant. The solubilized IntF-GST was bound to
glutathione-Sepharose 4B beads (Pharmacia) and purified by
electroelution after electrophoresis on a 10% separating sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel.
Expression of AfRho1p and purification of recombinant Rho1p.
AfRHO1 cDNA with a CCAAG Kosak consensus sequence located
immediately upstream of the ATG translation start and a six-His tag
immediately downstream of the ATG start was obtained by PCR and was
cloned in the P. pastoris intracellular expression vector pPIC3 at the BamHI/EcoRI site. Recombinant Rho1p
was expressed in the P. pastoris SMD1168 strain. Recombinant
yeasts were grown until saturation in buffered minimal glycerol
medium-yeast extract (BMGY) (Invitrogen), and after 48 h of
expression in the presence of methanol (BMMY) (Invitrogen), the
P. pastoris yeasts were recovered by centrifugation, washed
with water, and disrupted in a Braun MSK homogenizer using glass beads
of 0.5-mm diameter for 5 min in a 50 mM sodium phosphate buffer, pH
7.5, containing 1 mM phenylmethylsulfonyl fluoride and 3% glycerol
(PPG). Cell walls were removed by centrifugation for 10 min at
4,000 × g. The intracellular extract was centrifuged at 36,500 × g for 1 h. The supernatant (Rho1Sup)
was stored at 
80°C, and the pellet was resuspended in PPG
supplemented with 2%
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS).
After 30 min of incubation at 4°C, the extract was centrifuged at
36,500 × g for 1 h, and the solubilized proteins
(Rho1PSo1) were stored at 80°C. Recombinant Rho1p from Rho1Sup and
Rho1Pso1 was purified on Ni2+ columns (Invitrogen) and was
eluted with 50 mM histidine.
MMF of A. fumigatus.
A microsomal membrane
pellet was recovered after 1 h of centrifugation at
36,500 × g at 4°C of the membrane extract as
described previously (2) and was resuspended in EB (50 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 0.2 M NaF, 1 M sucrose). This extract was
stored under liquid nitrogen and named the microsomal membrane fraction (MMF).
Purification of the glucan synthase complex by product
entrapment.
The A. fumigatus glucan synthase complex
was purified by product entrapment by following the procedure described
by Kelly et al. (23), with the following modifications:
(i) solubilization of MMF extract was carried out at room temperature
for 30 min with 0.5% polyoxyethylene ether W1 (W1) (Sigma) in EB
containing 20 µM GTP-
-S, and (ii) two incubations with 2 mM UDP
glucose at 4°C for 10 min were performed. Solubilization of the
proteins from the product entrapment pellet (PE pellet) was done in EB containing 0.1% W1, and centrifugation was done at 60,000 × g for 15 min at 4°C and was repeated once. Combined
supernatants were referred to as the product entrapment fraction (PE).
Nonspecific binding of W1-solubilized proteins to (1-3) glucan was
assessed by incubating the W1-solubilized MMF extract for 30 min at
25°C with (1-3) glucan from the PE pellet digested with
proteinase K (Sigma) (4 mg for 45 min at 56°C) to remove all proteins
associated with the neosynthesized glucan. All fractions were analyzed
by SDS-PAGE (25) after having dissolved the samples in
reducing denaturing 4× SDS electrophoresis buffer at room
temperature without heating.
PE proteins were identified by in-gel digestion after separation on
SDS-8% PAGE and Coomassie blue staining followed either by separation
of the resulting peptides by liquid chromatography and Edman sequencing
as described earlier (4) or by matrix-assisted laser
desorption ionization-time of flight (MALDI-TOF) analysis and tandem
mass spectrometry (MS). For MS, proteins were digested by trypsin in a
Progest robot (Abimed). After extraction, peptides were desalted on
Ziptip micro columns (Millipore), and eluates either were mixed with
MALDI matrix (saturated solution of dihydroxybenzoic acid) on the
MALDI-TOF target of the Voyager DE-STR mass spectrometer (Applied
Biosystems) or were infused by using the nanoelectrospray source kit
(Protana A/S) for the Finnigan TSQ 7000 (Thermoquest). MALDI-TOF (MS)
spectra were obtained by using an accelerating voltage of 21 kV in the
reflector mode. MS-MS was done on doubly charged protonated molecules
using argon (2.5 millitorr) as the collision gas at 20 to 38 eV.
MALDI-TOF (MS) and MS-MS data were matched against the National Center
for Biotechnology database using the MS-Fit and MS-Edman programs,
respectively (assembled into the Protein Prospector package, P. R. Baker and K. R. Clauser, http://prospector.ucsf.edu), over an
intranet connection.
ADP-ribosylation assay.
Ten microliters (50 to 100 µg) of
MMF, solubilized in 0.3% CHAPS as described by Beauvais et al.
(2), was incubated for 60 min at 37°C in the presence of
a solution containing 20 µM GTP, 7.5 µM
[32P]NAD+ (1.25 µCi), and 15 ng of
C3 exoenzyme from Clostridium botulinum (Biomol)
in a 60 mM HEPES buffer (pH 8) containing 1.5 mM MgCl2 and
1.5 mM 5'-AMP followed by SDS-PAGE with a 12% separating gel and by autoradiography.
Antibodies.
Purified recombinant Rho1 protein from Rho1Sup
(420 µg) produced by P. pastoris and the IntF-GST fusion
protein produced by E. coli (300 µg) and purified as
described above were used as antigens to raise anti-AfrRho1p and
anti-IntF antibodies, respectively, in rabbits (27). The
presence of specific antibodies in the animals was verified by Western
blotting by using the ECL chemiluminescence detection method of
Amersham. Preimmune rabbit serum was collected and used as a control.
Murine anti-(1-3) glucan monoclonal antibody was from Biosupplies
Australia (Victoria, Australia).
Localization of AfFks1p and (1-3) glucan.
Immunolocalization of AfFks1p was attempted on germ tubes.
Permeabilization and immunofluorescence studies were performed basically as described by Harris et al. (16), with the
following modifications. Coverslips with germ tubes were incubated in
phosphate-buffered saline (PBS) containing 5% goat serum (Sigma)
(PBS-goat serum) for 1 h at room temperature (RT) before
incubation in the anti-IntF or preimmune rabbit antisera diluted 1/20
for 1 h at RT. After being washed, labeling was performed using a
goat anti-rabbit fluorescein isothiocyanate conjugate diluted 1/50
(Sigma) for 1 h at RT. Antisera and fluorescein isothiocyanate
conjugates were diluted in PBS-goat serum, and all washings were done
in PBS-goat serum. After the final PBS washing, germ tubes were
observed under a fluorescent microscope with an excitation filter of
450 to 490 nm.
Localization of (1-3) glucan was done by incubating germ tubes
produced in liquid Sabouraud medium for 10 h at 37°C with aniline blue Water Blue (Fluka) at a concentration of 0.1 mg/ml in a
phosphate buffer (50 mM, pH 8.5) for 1 h at RT.
Nucleotide sequence accession numbers.
DNA sequences of
A. fumigatus FKS1, RHO1, RHO2, RHO3, and RHO4 are
available in the GenBank database under the accession numbers U79728,
AY007297, AY007298, AY007299, and AY007300, respectively.
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RESULTS AND DISCUSSION |
Cloning of A. fumigatus FKS1 and production of
recombinant Fks1p fragment.
The genomic DNA sequence of the
FKS1 optical reading frame (ORF) of A. fumigatus
was 5,813 bp long and was interrupted by two introns located at the N
terminus (nt 138 to 185) and the C terminus (nt 5466 to 5521). A TATA
transcription sequence was found 233 nt upstream of the ATG site.
AfFKS1 encoded a predicted protein of 1,903 amino acids with
an estimated molecular size of 218 kDa and a pI of 8.17. The amino acid
sequence of AfFKS1 was highly similar to other
FKS genes present in the databases (37).
Interestingly, it was almost identical to the A. nidulans FKS gene, with 90% amino acid identity, identical intron
positions, and the presence of the two domains D1 and D2 in the
conserved internal hydrophilic fragment homologous (44 and 29%
identity, respectively) to the catalytic subunit of bacterial cellulose synthase of Acetobacter xylinum (accession number SP19449)
(23). As with the other Fksp proteins, AfFks1p predicts an
integral membrane protein with a cytoplasmic N terminus, has 16 transmembrane domains, and contains the putative UDP glucose binding
consensus sequence RXTG at the C terminus (AfFks1p, RITG: amino acids
[aa] 1565 to 1568) (20, 23, 29, 30, 38).
Southern analysis of genomic DNA isolated from A. fumigatus
showed that only a single band hybridized to the HindIII
1.1-kb fragment of AfFKS1 under low stringency (data not
shown), suggesting the absence of homologs of AfFKS1. A
similar situation was found in Cryptococcus neoformans,
where only a single FKS gene was found (38).
Disruption of the FKS1 gene was also attempted in A. fumigatus. Transformation of A. fumigatus was performed
with a pBluescript SK(+) plasmid containing the AfFKS1 gene
interrupted by the pyrG gene or the phleomycin resistance
gene. Among the 105 transformants tested, none showed the insertion of
the disrupted gene at the AfFKS1 locus (data not shown),
whereas disruption of more than 10 different nonessential genes in our
laboratory using the same markers and transformation protocols always
resulted in gene disruption at the correct locus, with double crossing
over for 5 to 10% of the transformants (8, 9, 21; S. Paris, unpublished data; I. Mouyna, unpublished data). The essentiality
of the FKS1 gene was recently confirmed (A. Firon, M. C. Grosjean-Cournoyer, A. Beauvais, and C. d'Enfert, Abstr. 21st Fung.
Genet. Conf. Asilomar, abstr. 412, 2001) using a strategy previously
used with A. nidulans (3) and adapted to
A. fumigatus. The essentiality of the FKS gene
was also shown in S. cerevisiae when FKS1 and
FKS2 were both disrupted (29).
Attempts to produce the entire recombinant protein AfFks1p using the
baculovirus expression system failed because transcription in the
insect cell resulted in the production of a truncated mRNA (data not
shown). All attemps to obtained heterologous expression of Fksp have,
indeed, failed to date (M. Kurtz, personal communication). It was then
decided to express part of the protein in E. coli. The
conserved hydrophilic internal fragment (IntF) (aa 841 to 1265) of the
A. fumigatus amino acid sequence was selected to be
expressed in E. coli. A GST fusion protein with a molecular size of 74 kDa was produced which released a polypeptide of 48 kDa
after human thrombin (Sigma) digestion (0.25 U of thrombin in PBS
containing 2.5 mM CaCl2 for 1 h at RT), corresponding
to the expected molecular size of IntF (Fig.
1). This protein was used to raise
anti-AfFks1p antibodies in rabbit serum (anti-IntF antiserum). The
N-terminal fragment (aa 1 to 387) could also be expressed in E. coli, but in contrast, expression of the C terminus was
unsuccessful (aa 1441 to 1904) (data not shown).
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FIG. 1.
Expression of IntF in E. coli (A) Coomassie
blue staining of proteins after SDS-PAGE with a 10% separating gel.
Lane 1, IntF-GST fusion protein; lane 2, IntF polypeptide after
thrombin digestion (note that the digestion was incomplete with two
polypeptides corresponding to the undigested IntF-GST and IngF). (B)
Immunolabeling of IntF-GST and IntF with anti-IntF antibodies (1/1,000
dilution). Lanes 1 and 2 correspond to lanes 1 and 2 of panel A. No
labeling was seen when lanes 1 and 2 of panel A were incubated with
preimmune sera (not shown). The numbers at the left of each panel are
molecular sizes in kDa.
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Cloning of A. fumigatus RHO genes and production of
recombinant AfRho1p.
In A. fumigatus, four
RHO genes (RHO1 through RHO4) were
cloned. However, only RHO1 was studied, because it showed
the highest amino acid homology to RHO1 of the yeast
S. cerevisiae (Fig. 2).
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FIG. 2.
Phylogenetic dendrogram of Rho proteins of A. fumigatus (AfRho1, AfRho2, AfRho3, AfRho4), S. cerevisiae (ScRho1, ScRho2, ScRho3, ScRho4), C. albicans (CaRho1), and S. pombe (SpRho1, SpRho2).
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The RHO1 ORF was 947 bases long. The ORF was interrupted by
four introns spanning nt 69 to 164, 193 to 314, 513 to 605, and 741 to
776. The cDNA (600 nt) coded for a protein of 21.5 kDa. The amino acid
sequence of AfRho1p was highly homologous to that of Rho1p of
Schizosaccharomyces pombe, S. cerevisiae, and Candida albicans, showing 85, 79, and 60% identity, respectively (Fig. 3). Amino acid sequences of Rho1p of
A. fumigatus, S. cerevisiae, C. albicans, and S. pombe presented similar motifs for the GTP binding and GTP
hydrolysis sites (Fig. 3). The recognition sequence for prenylation,
CTIL, was also present at the C-terminal end of the protein but was
different from those of yeast (Fig. 3) (18). In AfRho1p,
the first aliphatic amino acid (a1) of the yeast consensus
sequence Ca1a2L was replaced by the polar amino acid threonine. The same modification was found in AfRho3p, whereas in
AfRho2p the sequence was identical to the one found in yeast. AfRHO4 was not fully sequenced.
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FIG. 3.
Alignment of Rho1 proteins of A. fumigatus
(AfRho1), S. pombe (SpRho1), S. cerevisiae
(ScRho1), and C. albicans (CaRho1). GTP binding and GTP
hydrolysis domains are indicated by stars and dots, respectively.
Geranyl-geranylation domains of each Rho1p are underlined.
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Recombinant AfRho1p was expressed in P. pastoris. A
polypeptide of 21 kDa was found in the cell lysates (Fig.
4). This 21-kDa polypeptide was recovered
in the cellular extract both in the Rho1Sup and in the Rho1PSo1
fractions. Recombinant Rho1p from Rho1Sup purified from a
Ni2+ column (Fig. 4) was used to raise anti-AfrRho1p
antibodies in rabbits. The antiserum specifically reacted with the
recombinant AfRho1p (Fig. 4), whereas control membranes of P. pastoris were completely negative, indicating that the antiserum
did not recognize Rho1p of P. pastoris (data not shown).
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FIG. 4.
Recombinant Rho1p (rRho2p). P. pastoris
proteins were separated by SDS-PAGE with a 12% separating gel followed
by Coomassie blue staining of molecular size standards in kDa (lane 1),
cell lysate of control P. pastoris without rRho1p (lane 2),
cell lysate of P. pastoris expressing rRho1p after methanol
induction (lane 3), and purified rRho1p from Rho1Sup (lane 4) and
Rho1PSol (lane 5) fractions of recombinant P. pastoris.
Lanes 6 and 7, immunolabeling of rRho1p from lanes 3 and 4, respectively, with the antiserum against the recombinant AfRho1p
(1/15,000 dilution). No labeling was seen when the lysate shown in lane
2 was incubated with anti-AfrRho1p antibodies (not shown).
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Analysis of the protein complex associated with newly synthesized
(1-3) glucans.
Incubation of the W1-solubilized membrane
proteins of A. fumigatus with UDP glucose resulted in the
isolation of a protein mixture which coprecipitated with the (1-3)
glucan synthesized and which still displayed a glucan synthase
activity. In A. fumigatus, W1 detergent was an activator
(1.5- to 3-fold increase in glucan synthase activity in the presence of
0.5% W1), whereas it inhibited the glucan synthase activity in
A. nidulans (23). The product entrapment method
achieved a 240-fold purification in one cycle of product entrapment
(Table 1). Another cycle of entrapment did not result in further purification (data not shown). The proteins associated with (1-3) glucan were investigated by immunoblotting using anti-IntF and anti-AfrRho1p antisera and by mass spectrometry.
Antiserum against IntF strongly reacted with a protein in the product
entrapment extract, migrating at 180 kDa (p180, Fig. 5A, lanes 4 and 6). MALDI-TOF (MS) and
nanoelectrospray MS-MS confirmed that this band corresponded to
AfFks1p. Immunolabeling of a membrane preparation prior to product
entrapment with the same anti-IntF antiserum was negative even at high
concentrations (1/40 dilution) (data not shown) (Fig. 5A, lanes 3 and
6). Similar enrichment of Fks1p was obtained in S. cerevisiae (33). Antiserum directed against
anti-AfrRho1p reacted with a 21-kDa protein with the expected molecular
size of AfRho1p in a membrane extract, and this protein was highly
enriched in product entrapment (Fig. 5B). The identity and
functionality of the 21-kDa protein were confirmed by ADP-ribosylation,
which was specific for the Rho1 protein (5), using the
C3 exoenzyme and [32P]NAD+ on
membrane-solubilized proteins from A. fumigatus (Fig.
6).
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FIG. 5.
Analysis of the (1-3) glucan synthase complex after
product entrapment. (A) Coomassie blue staining of proteins (15 µg of
protein per lane) separated by SDS-PAGE with an 8% separating gel of
molecular size standards in kDa (lane 1), MMF (lane 2), W1-solubilized
MMF (lane 3), and PE (lane 4). Inserts show immunoblotting of PE (lane
5) and MMF (lane 6) with anti-IntF (1/1,000 dilution) and PE (lane 7)
with an anti-(1-3) glucan antibody (1/1,000 dilution). MMF remained
negative after incubation with anti-IntF diluted 1/40 (not shown). (B)
Immunoblotting with anti-AfrRho1p antibodies (1/15,000 dilution) of MMF
(lane 1), solubilized MMF (lane 2), and PE (lane 3). A total of 2.6 µg of protein per lane after separation of the proteins by SDS-PAGE
with a 12% separating gel was used.
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FIG. 6.
ADP-ribosylation of Rho1p in A. fumigatus.
Lane 1, molecular size standards in kDa; lane 2, Coomassie blue
staining of CHAPS-solubilized MMF proteins separated by SDS-PAGE with a
12% separating gel; lane 3, autoradiography of CHAPS-solubilized MMF
incubated with 7.5 µM [32P]NAD+ and 15 ng
of C3 exoenzyme for 1 h at 37°C followed by SDS-PAGE
(12% separating gel).
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Two other proteins of large molecular size with apparent sizes of 100 and 160 kDa were enriched in the PE (p100 and p160, Fig. 5A, lane 4).
These two proteins were only enriched when the glucan synthase was
functional. Indeed, when a solubilized membrane preparation was
incubated with (1-3) glucan purified from the PE pellet treated
with protease, the 100- and 160-kDa (as well as AfFks1p and AfRho1p)
proteins were not found in the (1- 3) glucan pellet recovered by
centrifugation, indicating that the enrichment of these proteins did
not result from their nonspecific binding to neosynthesized (1-3)
glucan. A similar electrophoretic gel pattern was obtained during PE
purification of A. nidulans glucan synthase
(23). Two peptide sequences, TVYFGEIGEK and KXGEMLVVLGRP,
were obtained for the 160-kDa protein. They matched with the ABC
transporter pmr1 of Penicillium digitatum
(accession number AB010442), which showed approximately 40% similarity with two ABC bacterial transporters, chrA in
Agrobacterium tumefaciens (13) and
ndrA in Rhizobium meliloti (37),
which are involved in the export of (1-2) glucans in these
organisms. This 160-kDa protein reacted with an anti-(1-3) glucan
antibody, suggesting that this protein was bound to glucan (Fig. 5A,
lane 7). Peptides DNLGRKTRSKA, DSERLIHG, DEYTALNRYL, AREILSYN,
KADEFARV, KYTPFDG, and KYQVVEMLQQ were obtained for the 100-kDa
protein and were matched with a plasma membrane H+-ATPase
of A. nidulans (accession number AF043332), migrating at 110 kDa in the PE extract SDS-PAGE gel from A. nidulans
(23).
Cellular localization of glucan synthase.
When aniline blue, a
fluorochrome specific for (1-3) glucans (6), was added
to growing germ tubes, the apex of the germ tube was the point most
intensively labeled by the aniline blue, indicating that the newly
synthesized (1-3) glucan was produced at the apex of the germ tube
(Fig. 7). The apex also was positively labeled with the anti-IntF antiserum. This result showed that AfFks1p
was localized at the apical growing region of the mycelium (Fig. 7).
Similar results were found in yeasts where Fksp was localized in the
bud (7).
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FIG. 7.
(A and B) Immunofluorescence of a germ tube of A. fumigatus labeled with anti-IntF antibodies and goat anti-IgG
fluorescein. (A) Anti-IntF antiserum, arrow indicates the apex of the
germ tube; (B) preimmune serum. (C and D) Labeling of a germ tube of
A. fumigatus with aniline blue fluorochrome. (C)
Epifluorescence; (D) transmitted light.
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We thank H. Chaabihi (Quantum Biogene) and his collaborators for
the construction of the expression AfFKS1 vector for the baculovirus expression system, J.-P. Le Caer at the Ecole
Supérieure de Physique et de Chimie Industrielle, Paris, France,
for conducting mass spectrometry experiments, and C. Fudali from
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Abstract
Introduction
Present address: Swammerdam Institute for Life Science, University
of Amsterdam, Amsterdam, The Netherlands.
