From the Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, 93040 Regensburg, Germany
Received for publication, December 10, 2002, and in revised form, January 23, 2003
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ABSTRACT |
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Protein O-mannosyltransferases (PMTs)
initiate the assembly of O-mannosyl glycans, an essential
protein modification. Since PMTs are evolutionarily conserved in fungi
but are absent in green plants, the PMT family is a putative target for
new antifungal drugs, particularly in fighting the threat of
phytopathogenic fungi. The PMT family is phylogenetically classified
into PMT1, PMT2, and PMT4 subfamilies, which differ in protein
substrate specificity. In the model organism Saccharomyces
cerevisiae as well as in many other fungi the PMT family is
highly redundant, and only the simultaneous deletion of PMT1/PMT2 and
PMT4 subfamily members is lethal. In this study we analyzed the
molecular organization of PMT family members in S. cerevisiae. We show that members of the PMT1 subfamily (Pmt1p and
Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and
Pmt3p) and that Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes represent the
predominant forms. Under certain physiological conditions, however,
Pmt1p interacts also with Pmt3p, and Pmt5p with Pmt2p, suggesting a
compensatory cooperation that guarantees the maintenance of
O-mannosylation. Unlike the PMT1/PMT2 subfamily members,
the single member of the PMT4 subfamily (Pmt4p) acts as a homomeric
complex. Using mutational analyses we demonstrate that the same
conserved protein domains underlie both heteromeric and homomeric
interactions, and we identify an invariant arginine residue of
transmembrane domain two as essential for the formation and/or
stability of PMT complexes in general. Our data suggest that
protein-protein interactions between the PMT family members offer a
point of attack to shut down overall protein
O-mannosylation in fungi.
Protein O-mannosylation is an evolutionarily conserved
protein modification of fundamental importance in many eukaryotes. In
yeasts and fungi, the attachment of O-linked mannosyl
residues to proteins of the secretory pathway is essential for cell
viability (1). In particular it is indispensable for cell wall
integrity and normal cellular morphogenesis (2-4). Impairment of
O-mannosylation also affects the stability, localization,
and/or proper function of individual proteins (5-10). Furthermore,
aberrant O-mannosylation can interfere with the retrograde
transport of misfolded proteins across the membrane of the endoplasmic
reticulum (ER)1 (11).
O-mannosylation is not only important in yeast, but also in
mammals. It was recently shown that in humans, O-mannosyl
glycosylation represents a new pathomechanism for muscular dystrophy
and neuronal migration disorders (12, 13).
In yeast and fungi, O-mannosylation is initiated in the
lumen of the ER by an essential family of protein
O-mannosyltransferases (PMTs). These
enzymes catalyze the transfer of mannose from dolichyl phosphate-activated mannose (Dol-P-Man) to serine or threonine residues
of secretory proteins (2). In Saccharomyces cerevisiae, a
total of seven PMT family members (Pmt1-7p) have been identified, which share almost identical hydropathy profiles that predict the PMTs
to be integral membrane proteins with multiple transmembrane domains.
Pmt1-6 proteins feature an overall protein sequence identity of
57.5%. Pmt7p is less conserved. Protein
O-mannosyltransferase activity has been demonstrated for
Pmt1-4p and Pmt6p (14). Aside from S. cerevisiae PMTs,
orthologues are known from many other yeast and fungi, for example,
Candida albicans (CaPMT1-2 and
CaPMT4-6) and Schizosaccharomyces pombe
(SpPMT1, SpPMT3, and SpPMT4) (Refs. 3
and 4).2 Moreover, PMT
homologues have been also identified in many multicellular eukaryotes
such as Drosophila melanogaster, mouse and, humans (15-17).
Despite their evolutionarily conservation in fungi and throughout the
animal kingdom (with the exception of Caenorhabditis elegans), PMTs are not present in green
plants.2 This makes the PMT family in fungi
especially attractive as a target for the development of new antifungal
drugs in order to combat phytopathogenic fungi.
The protein O-mannosyltransferases can be divided into three
subfamilies: PMT1, PMT2, and PMT4, which include transferases closely
related to S. cerevisiae Pmt1p, Pmt2p, and Pmt4p,
respectively (17, 18). Members of the PMT1 and PMT2 subfamilies show
marked similarities and distinctions from PMT4 subfamily members.
First, all PMT family members share three conserved sequence motifs
but, these show significant variations between PMT1/PMT2 and PMT4
subfamily members (18). Second, the PMT1/PMT2 and PMT4 subfamilies use distinct acceptor protein substrates in vivo (9, 14). Third, in fungi the PMT1/PMT2 subfamily is highly redundant, whereas the PMT4
subfamily has only one representative per species (17, 18).
Among the PMT family members, Pmtp1 from S. cerevisiae has
been most extensively characterized. Pmt1p is an integral ER membrane glycoprotein with seven transmembrane-spanning domains (19). Its N
terminus faces the cytoplasm whereas the C terminus faces the lumen of
the ER. Two major hydrophilic domains that are located between
transmembrane spans one and two (loop 1) and transmembrane spans five
and six (loop 5), respectively, are oriented toward the ER lumen and
are essential for Pmt1p activity (18, 19). The replacement of invariant
amino acid residues in these regions suggested that these segments are
involved in the recognition and/or binding of protein substrates and/or
catalysis (18). Comparison of PMTs from different organisms defined
highly conserved peptide motifs present in loop 5 (18), which are also
found in IP3 and ryanodine receptors. Their common function
is unknown (20). Pmt1p forms a heteromeric complex with Pmt2p in
vivo, and this complex formation is essential for maximal
mannosyltransferase activity (18, 21). N- and C-terminal regions of
Pmt1p are involved in Pmt1p-Pmt2p interactions (18). Other than Pmt1p, very little is known about the molecular organization of the rest of
the O-mannosylation machinery.
In the present study we analyzed the molecular assembly of the PMT
family in yeast. We demonstrate that complex formation is of general
validity for all members of the PMT family in S. cerevisiae.
Strikingly, members of the PMT1 subfamily form specific heteromeric
complexes with members of the PMT2 subfamily in vivo, while
Pmt4p acts as a homomeric complex. Despite the differences between
PMT1/PMT2 and PMT4 complexes, we show that the same rules and residues
govern Pmtp protein-protein interactions.
Strains and Plasmids--
The S. cerevisiae strains
are listed in Table I. Yeast strains were
grown under standard conditions and transformed following the method of
Gietz et al. (27) with the yeast shuttle vectors pRS423
(28), YEp352 (29), pSB53 (19), pSB56 (18), PMT2-YEp352 (23), pVG13
(18), pSB114 (18), and the plasmids listed below. Standard procedures
were used for all DNA manipulations (30). All cloning and
transformations were carried out in Escherichia coli host
SURE®2 (Stratagene). PCR fragments were routinely checked
by sequence analysis. Oligonucleotide sequences are available upon
request.
Plasmid pVG80 (PMT2HA)--
A SalI site
was introduced downstream of the PMT2 coding region by
cloning a 2.96-kb PstI/HindIII fragment from
PMT2-YEp352 (23) into pBluescript SK+
(Stratagene) digested with the same enzymes. From the resulting plasmid
pVG70 a 2.97-kb PstI/SalI fragment was isolated
and cloned into YEp352 (cut with PstI and SalI),
resulting in plasmid pVG76. A total of six copies of the hemagglutinin
(HA) epitope were fused to the C terminus of PMT2 by
recombinant PCR (31). Two separate PCR products that overlap in
sequence were produced. One was amplified by PCR on pVG76 with the
oligonucleotides vg65 and vg66, the other on plasmid pHA-kanMX (gift of
U. Schermer) with the oligonucleotides vg67 and vg68. The overlapping,
primary PCR products were combined into one longer product using
oligonucleotides vg65 and vg68. The resulting 730-bp fragment was
cloned into pGEM T-easy (Promega). A 695-bp
BglII/SalI fragment of the resulting plasmid
pVG78 was subcloned into pVG76 (cut with BglII and
SalI). DNA sequence analysis of the resulting plasmid pVG80
(PMT2HA) was performed. In the course of this
analysis, we realized a discrepancy between the PMT2
sequence we obtained and the yeast data base entry
(GenBankTM accession no. AAC04934). To verify the
PMT2 sequence, we amplified a 664-bp genomic DNA fragment of
PMT2 from the yeast strains S288c, BY4742, W303-1A, and
SEY6210 using the oligonucleotides vg63 and vg69. PCR products were
cloned into pGEM T-easy, and several independent clones were sequenced.
These analyses showed that in contrast to the data base entry, the
PMT2 open reading frame contains three additional base pairs
(bp +400 to +429 is tgggacttccCttctggGGaaatttaccca; additional bases in
capital letters). The insertions result in the predicted amino acid
sequence of Pro-Ser-Gly instead of Leu-Leu at position 137 of Pmt2p.
Plasmid pJK4-B1 (PMT4FLAG)--
A copy of the FLAG
sequence (32) was obtained by annealing oligonucleotides oligo211 with
oligo212. The annealed oligo pair features BamHI and
NotI overhang sequences. The FLAG sequence was joined by a
three-piece ligation with a 0.7-kb SacI/NotI
fragment (isolated from plasmid SAP/EN, Ref. 33) that contains the
yeast plasma membrane ATPase terminator (34) and the yeast shuttle vector pRS423 (digested with SacI and NotI)
resulting in plasmid pRS423/TER/FLAG. The PMT4 promoter and
coding region (bp Plasmid pVG37 (PMT4 Plasmid pVG45 (PMT4 R142EFLAG)--
An arginine
residue at position 142 of Pmt4pFLAG was exchanged for a
glutamate by site-directed mutagenesis (GeneEditorTM, Promega) using
the oligonucleotide vg30 and plasmid pVG43 (pUC18, containing bp +24 to
+1165 of PMT4). Mutations were confirmed by DNA sequence analysis of the resulting plasmid pVG44. A 476-bp MunI
fragment of pVG44 was cloned into pVG42 (pUC18, containing bp +307 to
+1165 of PMT4) digested with the same enzyme. From the
resulting plasmid pVG69 a 1.87-kb SphI/HpaI
fragment was isolated and cloned into pJK4-B1 resulting in pVG45.
Plasmid pVG36 (PMT1HAR138K)--
Amino acid Arg-138
of Pmt1pHA was changed to lysine by site-directed
mutagenesis using the oligonucleotide vg16 and plasmid pVG26 (pUC19,
containing bp +61 to +537 of PMT1). Mutations were confirmed
by DNA sequence analysis. A 335-bp NcoI/BsrGI
fragment of the resulting plasmid pVG29 was cloned into plasmid pVG20
(pUC19, containing a 990-bp EcoRI/PstI fragment
of pSB56, Ref. 18) cut with NcoI/BsrGI. From the
resulting plasmid pVG28 a 594-bp PmlI/BsrGI fragment was isolated and cloned into pSB56 to generate pVG36.
Production of Polyclonal Anti-Pmt3-6p Antibodies in
Rabbits--
Rabbits were immunized with recombinant fusion proteins
consisting of glutathione S-transferase, and the aa Met-1 to
Arg-78 of S. cerevisiae Pmt3p, aa Met-1 to Ala-65 of Pmt4p,
aa Asp-10 to Thr-121 of Pmt5p, and aa Met-1 to Gln-85 of Pmt6p,
respectively. The corresponding DNA fragments were amplified by PCR
using genomic S. cerevisiae DNA as template and adapter
oligonucleotides oligo151/oligo152 for PMT3,
oligo106/oligo107 for PMT4, oligo143b/oligo144 for
PMT5, oligo147/oligo148 for PMT6. The respective
DNA fragments were combined with the glutathione
S-transferase sequence by EcoRI/BamHI subcloning into a pGEX-2TK expression vector (Amersham Biosciences). The fusion proteins were expressed in E. coli host BL21. The
recombinant proteins were excised from SDS-polyacrylamide gels and
injected into rabbits. Pineda Antikoerper-Service, Berlin, Germany,
performed immunizations. Antibodies were affinity-purified by binding
to nitrocellulose derivatized with the glutathione
S-transferase fusion protein (35).
Preparation of Crude Membranes--
Crude membranes were
isolated as described (18).
Immunoprecipitation--
Sodium deoxycholate extracts were
prepared as described (18). Immunoprecipitation of Pmt1pHA,
Pmt2pHA and Pmt3-6p was performed using 300 µl of sodium
deoxycholate extract made from 109 cells of the appropriate
yeast strains. Pmt1pHA was immunoprecipitated with 10 µl
of anti-HA monoclonal antibody covalently coupled to protein
A-Sepharose (16B12, Babco) for 1-2 h at 4 °C. To precipitate
Pmt3-6p, affinity-purified polyclonal anti-Pmt3-6p antibodies were
covalently coupled to protein A-Sepharose (Pmt3-6p beads) as described
(36). Pmt3-6p were immunoprecipitated with 20-50 µl of Pmt3-6p
beads for 1-2 h at 4 °C. Immunoprecipitates were washed four times
with 1 ml of cold lysis buffer (20 mM Tris-HCl, pH 7.5, 140 mM NaCl, 0.3 mM MgCl2,
10% (v/v) glycerol, 0.35% sodium deoxycholate, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM
benzamidine, 0.25 mM
N
Pmt4pFLAG and FLAG-tagged Pmt4p mutant proteins were
solubilized from crude membranes, prepared as described in Ref. 18
using 500 µl of solubilization buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 0.3 mM MgCl2,
0.5% Triton X-100, plus protease inhibitors) by vortexing for 30 min
at 4 °C. The suspension was clarified by centrifugation for 30 min
at 20,000 rpm (Sorvall SS34 rotor) to obtain the Triton extract.
FLAG-tagged proteins were immunoprecipitated for 1-2 h at 4 °C from
300 µl of Triton extract, using 30 µl of anti-FLAG M2 affinity Gel
(Sigma). Precipitates were washed four times with 1 ml of cold
solubilization buffer, once with 1 ml of Tris-buffered saline and
resuspended in 20 µl of 3× SDS-sample buffer.
Blue Native Polyacrylamide Gel Electrophoresis--
Yeast cells
were grown to 2-4 × 107 cells/ml in YPD medium (37).
A total of 1010 cells were harvested and crude membranes
were isolated (18) using a modified buffer (50 mM Tris-HCl,
pH 7.5, 15% (v/v) glycerol, plus protease inhibitors). Membranes were
resuspended in 1 ml of Nonidet P-40 solubilization buffer (50 mM Tris-HCl, pH 7.5, 0.5% Nonidet P-40, 750 mM
aminocaproic acid, 15% (v/v) glycerol, plus protease inhibitors).
Membrane proteins were solubilized by vortexing for 20 min at room
temperature. The suspension was clarified by ultracentrifugation at
100,000 × g at 4 °C for 60 min. Samples (40 µg of
protein) were loaded onto polyacrylamide gradient gels. Just prior to
electrophoresis, Coomassie Brilliant Blue G (Serva; dissolved in 500 mM aminocaproic acid) was added from a 5% stock solution
to adjust to a detergent/Coomassie ratio of 4:1 (g/g). Blue native
polyacrylamide gel electrophoresis (BN-PAGE) was performed using a
polyacrylamide gradient from 4-13%, with a 4% stacking gel, as
described (38). 0.02% Nonidet P-40 was added to both polyacrylamide
gel and cathode buffer (50 mM Tricine, 15 mM
Bis-Tris). The cathode buffer also contained 0.02% Coomassie Brilliant
Blue G. This buffer was replaced after electrophoresis for 1 h at
80 V, 4 °C, by cathode buffer without Coomassie Blue. Electrophoresis was continued for ~6 h at 100 V, 4 °C. Gels were blotted onto nitrocellulose membranes using 20 mM Tris, 150 mM glycine, 20% MeOH, 0.02% SDS.
Chemical Cross-linking of Yeast Membrane Proteins--
2.0 × 109 yeast cells from a logarithmically growing culture
were harvested at 3,000 × g, 4 °C and washed once
with 20 ml of OPA buffer (50 mM boric acid/sodium
tetraborate, pH 8.0). The cell pellet was resuspended in 200 µl of
OPA buffer plus protease inhibitors. Crude membranes were prepared as
described (18). The membrane pellet was resuspended in 20 ml of OPA
buffer without protease inhibitors and centrifuged for 30 min at 20,000 rpm (Sorvall, SS34 rotor). Subsequently, membranes were resuspended in
500 µl of OPA buffer. For cross-linking, o-phtaldialdehyde
(OPA; Sigma) was added to final concentrations of 25-200
µM to 100 µl of membrane suspension and incubated in
the dark for 30 min at 25 °C. The reaction was quenched with 100 mM Tris-HCl, pH 6.8.
Western Blot Analysis--
Proteins were fractionated by
SDS-PAGE or BN-PAGE and transferred to nitrocellulose. Polyclonal
anti-Pmt1p (39) and anti-Wbp1p (40) antibodies were used at a dilution
of 1:2000, and anti-Pmt2p (21) at a dilution of 1:1000.
Affinity-purified antibodies anti-Pmt4p, anti-Pmt5p, and anti-Pmt6p
were used at a dilution of 1:2500; anti-Pmt3p at a dilution of 1:500.
The anti-HA (16B12, Babco) and anti-FLAG (M2, Sigma) monoclonal
antibodies were used at 1:8000 and 1:5000 dilutions, respectively.
Protein-antibody complexes were visualized by enhanced
chemiluminescence using the Amersham Biosciences ECL system.
In Vitro Dol-P-Man:Protein O-Mannosyltransferase
Assay--
Dol-P-Man:protein O-mannosyltransferase activity
was measured as described (14).
To uncover common principles that underlie the functionality of
Pmtps we investigated whether complex formation is a general feature of
the yeast PMT family members. We generated polyclonal antibodies that
specifically recognize S. cerevisiae Pmt3p (predicted molecular mass, 86.2 kDa), Pmt4p (predicted mass, 87.8 kDa), Pmt5p (predicted mass, 84.8 kDa), and Pmt6p (predicted mass, 87.9 kDa) in
wild-type yeast as shown by Western blot analyses (Fig.
1, lanes 1, 3,
5, and 7). Extracts from the corresponding
pmt deletion strains contained no cross-reactive material,
proving that the antibodies are highly specific (Fig. 1, lanes
2, 4, 6, and 8). We also created
epitope-tagged versions of Pmt2p (Pmt2pHA) and Pmt4p
(Pmt4pFLAG) (described under "Experimental
Procedures"). These tools enabled us to detect PMT complexes isolated
by coimmunoprecipitation, BN-PAGE, and chemical cross-linking.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Yeast strains
675 to +2286) was amplified from S. cerevisiae genomic DNA using oligonucleotides oligo213 and
oligo214. The PCR fragment was digested with BamHI and
SalI, and cloned into pRS423/TER/FLAG. Additional FLAG
sequences were cloned into the BamHI site of the resulting
plasmid pJK4, using the annealed oligonucleotide pair
oligo233/oligo234. In the resulting plasmid (pJK4-B1) four copies of
the FLAG epitope were fused to the C terminus of PMT4.
loop5)--
To remove amino acids (aa)
394-521 of Pmt4p, plasmid pJK4-B1 was digested with
PflMI/HpaI and religated using the adapter oligonucleotides vg23 and vg24.
-p-tosyl-L-lysine
chloromethyl ketone, 50 µg/ml TPCK, 10 µg/ml leupeptin, and
1 µg/ml pepstatin) and once with 1 ml of Tris-buffered saline.
Subsequently, precipitates were resuspended in 20 µl of 3× SDS
sample buffer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Specificity of polyclonal anti-Pmt3-6p
antibodies. Nonidet P-40 extracts (10 µg of protein) from yeast
wild-type strain SEY6210 (WT) and pmt3-6
deletion mutants (pmt3-6) were resolved on 8%
SDS-polyacrylamide gels and analyzed by Western blotting using
affinity-purified polyclonal anti-Pmt3p (lanes 1 and
2), anti-Pmt4p (lanes 3 and 4),
anti-Pmt5p (lanes 5 and 6), and anti-Pmt6p
(lanes 7 and 8) antibodies.
Members of the PMT1 Subfamily Interact in Pairs with Members of the
PMT2 Subfamily--
To characterize PMT complexes, we performed
coimmunoprecipitation experiments using a HA epitope-tagged version of
S. cerevisiae Pmt1p (Pmt1pHA, Ref. 18).
Pmt1pHA was expressed in a pmt1 deletion strain
and solubilized from crude membranes using Triton X-100 and sodium
deoxycholate. Pmt1pHA was immunoprecipitated from sodium
deoxycholate extracts with monoclonal anti-HA antibodies (see
"Experimental Procedures"). The immunoprecipitate and an aliquot of
the sodium deoxycholate extract were resolved on 8% SDS-polyacrylamide
gels and analyzed by Western blotting and sequentially probing the
blots with polyclonal antibodies to Pmt1p to Pmt6p (Fig.
2A, lanes 1 and
2). To ensure the specificity of the immunoprecipitation
reaction the same experiment was performed using strain
pmt1 expressing Pmt1p without the HA tag (Fig.
2A, lanes 3 and 4). As shown
previously (21) we confirmed that Pmt2p is the major interacting
partner of Pmt1p (Fig. 2A, lane 2). Moreover, a
weak signal for Pmt3p could be specifically detected in the
Pmt1pHA immunoprecipitate (Fig. 2A, compare
lanes 2 and 3). The amount of
coimmunoprecipitated Pmt3p was small, but this result was highly reproducible. In contrast, Pmt4p, Pmt5p, or Pmt6p could not be detected
(Fig. 2A, lane 2). Coimmunoprecipitation of
Pmt1pHA from pmt3 (Fig. 2B,
lane 4) and pmt2 (Fig. 2B, lane
6) deletion strains showed that Pmt1pHA interacts with
Pmt2p independently of Pmt3p and vice versa.
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To circumstantiate the existence of distinct Pmt1p-Pmt2p and
Pmt1p-Pmt3p complexes a HA-tagged version of Pmt2p was expressed in
strain pmt2 and coimmunoprecipitation experiments were
performed as described above. Fig. 2C shows that
Pmt2pHA specifically binds to Pmt1p but not Pmt3p, Pmt4p,
or Pmt6p (lane 2) corroborating that Pmt1p interacts with
Pmt3p independently of Pmt2p. In addition, in the Pmt2pHA
immunoprecipitate a weak signal for Pmt5p could be detected (Fig. 2C, lane 2). The amount of coimmunoprecipitated
Pmt5p was small; however, the result was specific (data not shown) and
highly reproducible. Furthermore, immunoprecipitation of
Pmt2pHA from a pmt1 deletion strain showed that
Pmt2pHA interacts with Pmt5p independently of Pmt1p (data
not shown).
So far our data indicated that in wild-type yeast Pmt1p and Pmt2p form a dominant protein complex. In addition, we found that Pmt1p also interacts with Pmt3p, and that Pmt2p interacts with Pmt5p. Next we addressed the question of which is the major interacting partner of Pmt3p. Pmt3p was immunoprecipitated from sodium deoxycholate extracts of wild-type and pmt3 mutant strains using polyclonal anti-Pmt3p antibodies (see "Experimental Procedures"). In the Pmt3p immunoprecipitate a small amount of Pmt1p, but not Pmt2p, Pmt4p, or Pmt6p was present (Fig. 2D, lane 2). Only Pmt5p was highly enriched when compared with the input material (Fig. 2D, compare lanes 1 and 2), demonstrating that Pmt3p predominantly interacts with Pmt5p. Again, the association between Pmt3p and Pmt5p was independent of other Pmt proteins (Fig. 2, A, C, and D and data not shown).
Summarizing, our data show that distinct Pmt1p-Pmt2p, Pmt1p-Pmt3p, Pmt5p-Pmt2p, and Pmt5p-Pmt3p complexes are present in S. cerevisiae. Of these, however, Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes represent the predominant forms.
The Pmt1p-Pmt3p and Pmt5p-Pmt2p complexes which we detected in our coimmunoprecipitation experiments are less abundant when compared with Pmt1p-Pmt2p or Pmt5p-Pmt3p complexes. To substantiate that the minor complexes are not formed artificially during coimmunoprecipitation, and to confirm that Pmt1p-Pmt2p and Pmt5p-Pmt3p represent the predominant PMT complexes in wild-type yeast, we performed BN-PAGE (38) that separates native protein complexes. Nonidet P-40 extracts derived from wild-type and pmt1-6 mutant strains were resolved on 4-13% polyacrylamide gels to separate native PMT complexes (see "Experimental Procedures"). Pmtp-containing complexes were detected by Western blots probed with polyclonal anti-Pmtp antibodies.
Analyses of Pmt1p-containing complexes showed that in Nonidet P-40
extracts from wild-type yeast two protein bands with an apparent mass
of ~140 kDa and ~310 kDa, respectively, were specifically detected
by anti-Pmt1p antibodies (Fig.
3A, compare lanes 1 and 7). The ~140-kDa band highly likely represents the
monomeric Pmt1p. The discrepancy in mass of ~140 kDa in BN-PAGE
versus 92 kDa in SDS-PAGE (19) is probably due to an
abnormal migration behavior of Pmt1p caused by the hydrophobic nature
of the protein, an unusual Coomassie Blue to protein ratio, and/or the
charge to mass ratio, which is variable in BN-PAGE (41). The formation
of Pmt1p homodimers was excluded by coimmunoprecipitation experiments
using Pmt1pHA and untagged Pmt1p (data not shown). In
addition to monomeric Pmt1p, specific Pmt1p-containing protein
complexes with an apparent molecular mass of ~310 kDa could be
detected (Fig. 3A, lane 1). The ratio of
monomeric Pmt1p to Pmt1p-containing high molecular weight complexes
varied to some extend in independent experiments (Fig. 3, A
and B, lane 1), which is highly likely due to
disaggregation of PMT complexes during solubilization. Protein
complexes with molecular masses of ~300-320 kDa were also detected
by polyclonal anti-Pmt2p, anti-Pmt3p and anti-Pmt5p antibodies (data
not shown).
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Assuming that in wild-type yeast in vivo Pmt1p-Pmt3p complexes are only present in minor amounts when compared with Pmt1p-Pmt2p, one would expect that in a pmt2 but not in a pmt3 deletion mutant the amount of the ~310-kDa complexes recognized by anti-Pmt1p antibodies should decrease dramatically. As shown in Fig. 3A (lanes 2 and 3), this is exactly what we observed. Nevertheless, in the absence of Pmt2p a very small amount of protein complexes with an apparent mass of ~310 kDa could be detected after raising the limit of detection of the Western analysis (Fig. 3B, lane 2). These complexes were not only recognized by anti-Pmt1p but also by anti-Pmt3p antibodies (data not shown). In pmt4-6 mutants Pmt1p-containing complexes were not affected (Fig. 3A, lanes 4-6). These data corroborate our finding that Pmt1p interacts individually with Pmt2p and Pmt3p; however, Pmt2p represents the major interacting partner. BN-PAGE also showed that Pmt3p-containing protein complexes vanished almost completely only in the absence of Pmt5p (data not shown), consistent with Pmt3p forming an abundant heteromeric complex with Pmt5p.
The amount of Pmt1p-Pmt3p and Pmt5p-Pmt2p complexes in wild-type yeast
appeared very minor. Therefore, the question comes up whether these
complexes are physiologically relevant. One possibility is that these
Pmtp complexes are mainly formed in the absence of their major
interacting partners to compensate partially for a lack of protein
O-mannosyltransferase activity. To investigate this
hypothesis, we analyzed pmt1pmt3 and pmt2pmt5
deletion mutant strains where the preferred interacting partners of
Pmt2p/Pmt5p, and Pmt1p/Pmt3p, respectively, are missing. Western
analysis of crude membranes isolated from a pmt1pmt3 mutant
showed that the amount of both Pmt2p and Pmt5p is increased when
compared with wild type (Fig.
4A, compare lanes 1 and 2). Accordingly, in the pmt2pmt5 mutant Pmt3p
is more abundant (Fig. 4A, compare lanes 1 and
3). The amount of Pmt1p is not obviously changed, suggesting that Pmt1p is not limiting (Fig. 4A, lane 3).
These observations suggested that in the absence of the favored
partners Pmt1p-Pmt3p and Pmt5p-Pmt2p complexes accumulate.
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To substantiate these data we analyzed Pmt5p-Pmt2p complex formation by chemical cross-linking using OPA. Under the conditions we applied (50 µM OPA) in wild-type yeast only upon overexpression of Pmt2p a protein complex with an apparent mass of ~152 kDa could be detected which is specifically recognized by polyclonal anti-Pmt5p (Fig. 4B, compare lanes 1 and 3) as well as anti-Pmt2p antibodies (data not shown). In contrast, Pmt5p-Pmt2p complexes could be easily detected in the pmt1pmt3 mutant even without overexpression of Pmt2p (Fig. 4B, lane 4).
To test whether the formation of Pmt5p-Pmt2p and Pmt1p-Pmt3p complexes results in increased O-mannosyltransfer, we determined in vitro O-mannosyltransferase activity in pmt1pmt3 and pmt2pmt5 mutant strains. The in vitro assay system we used preferentially detects O-mannosyltransferase activity of Pmt1p- and Pmt2p-containing complexes (Refs. 14 and 24, Table II). As shown in Table II in vitro O-mannosyltransferase activity is dramatically decreased in pmt1 and pmt1pmt2 mutants when compared with wild-type yeast. Western analysis of crude membranes revealed an equal abundance of Pmt3-6p in pmt1, pmt1pmt2 and wild-type strains (data not shown). In pmt1pmt3 and pmt2pmt5 mutants, in which Pmt5p-Pmt2p and Pmt1p-Pmt3p complexes are formed (see Fig. 4), in vitro O-mannosyltransferase activity is increased by 68.2% and 42.6%, respectively, when compared with pmt1pmt2 mutants (Table II).
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In summary, our data show that members of the PMT1 subfamily (Pmt1p and Pmt5p) form distinct complexes with members of the PMT2 subfamily (Pmt2p and Pmt3p), and that Pmt1p-Pmt2p and Pmt5p-Pmt3p pairs are the predominant forms. Under specific conditions Pmt1p-Pmt3p and Pmt5p-Pmt2p complexes can be formed to perpetuate O-mannosyltransfer.
Pmt4p Forms Homomeric Complexes in Vivo--
We could not detect
interactions between members of the PMT1 or PMT2 subfamily and Pmt4p,
the only member of the PMT4 subfamily. In agreement with these results,
no other Pmt proteins could be copurified when Pmt4p was precipitated
from sodium deoxycholate extracts from a yeast wild-type strain using
polyclonal anti-Pmt4p antibodies (data not shown). However, BN-PAGE
revealed the presence of larger Pmt4p complexes, which were not
influenced by the absence of any other PMT family member (data not
shown). We wished to determine whether these complexes were due to
Pmt4p-Pmt4p homotypic interactions or whether Pmt4p is associated with
other proteins. Therefore, an epitope-tagged version of Pmt4p was
constructed by fusing four copies of the FLAG epitope to the C terminus
of Pmt4p (see "Experimental Procedures"). Complementation of the temperature-sensitive phenotype of a pmt1pmt4 mutant by
Pmt4pFLAG proved that this construct is fully functional
in vivo (Fig. 7). To test for homotypic interactions,
PMT4FLAG was expressed in a S. cerevisiae wild-type strain and crude membranes were prepared.
Proteins were solubilized with Triton X-100 (Triton extract), and
immunoprecipitation of Pmt4pFLAG was performed using
monoclonal anti-FLAG antibodies covalently linked to protein
A-Sepharose. Immunoprecipitates were resolved on 8% SDS-polyacrylamide
gels. Wild-type Pmt4p and Pmt4pFLAG, which differ in
molecular mass by 4.8 kDa, were detected on a Western blot probed with
polyclonal anti-Pmt4p antibodies. As shown in Fig.
5A Pmt4pFLAG
specifically coimmunoprecipitates wild-type Pmt4p (compare lane 4 with lanes 5 and 6), indicating that Pmt4p
is present in homomeric complexes in vivo. Chemical
cross-linking experiments corroborated these results.
Pmt4pFLAG was expressed in a pmt4 deletion
strain, crude membranes were prepared, and cross-linking was performed
using OPA at final concentrations of between 25 and 100 µM. Proteins were resolved on 8% SDS-polyacrylamide gels
and analyzed by Western blotting using anti-Pmt4p antibodies as probe.
Fig. 5B (lane 2) shows that in the absence of
OPA, Pmt4pFLAG migrates with an apparent mass of ~90 kDa,
which is in agreement with a deduced mass of 92.6 kDa. Upon addition of
OPA, larger complexes with an apparent molecular mass of ~165 kDa
could be detected (Fig. 5B, lanes 3-5),
consistent with the formation of homodimeric Pmt4p complexes.
Summarizing, our data suggest that Pmt4p forms homomeric complexes;
however, the association with other smaller molecular weight molecules
cannot be ruled out completely.
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Conserved Protein Domains Underlie Heteromeric Pmt1p-Pmt2p and
Homomeric Pmt4p-Pmt4p Interactions--
Because in contrast to the
other PMT family members Pmt4p forms homomeric complexes, we asked
whether common principles underlie homomeric and heteromeric PMT
complex formation and/or stability. We previously showed that a large
hydrophilic endoplasmic reticulum-oriented segment of Pmt1p (loop 5, aa
294-586) is crucial for mannosyltransferase activity but not for
Pmt1p-Pmt2p complex formation (Ref. 18, see also Fig. 7). To test
whether the same may be the case for Pmt4p we deleted the large
predicted luminal loop 5 region of Pmt4pFLAG (aa 394-521,
Fig. 6A) and expressed the
internal deletion construct (loop5) in pmt4 mutant and
wild-type yeast strains. Pmt4p complex formation was assayed by
chemical cross-linking. Fig. 6B shows that in the presence
of OPA, larger complexes with an apparent mass of ~140 kDa (compare
lanes 1 and 2) can be detected in the pmt4 mutant strain in addition to monomeric
loop5.
Furthermore, when
loop5 and wild-type Pmt4p are expressed
simultaneously, additional complexes varying in size from ~156 to
~170 kDa appeared (Fig. 6B, lane 4). From these
data we conclude that Pmt4pFLAG
loop5 is able to
interact with itself as well as with wild-type Pmt4p. When
Pmt4pFLAG
loop5 is expressed in a temperature-sensitive
pmt1pmt4 mutant strain it does not restore the growth defect
at 35 °C (Fig. 7), indicating that
this large hydrophilic segment is essential for Pmt4p activity even
though it does not obviously affect Pmt4p dimerization. Our data show
that loop 5 domain of Pmt4p appears to behave in the same way as loop 5 of Pmt1p.
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|
For S. cerevisiae Pmt1p, amino acid residue Arg-138, located
in transmembrane domain two at the water-membrane interface, is
essential for the formation of heteromeric Pmt1p-Pmt2p complexes (Ref.
18, see also Fig. 6C, lane 3). In addition,
exchange of Pmt1p Arg-138 for alanine results in a complete loss of
mannosyltransferase activity (Ref. 18, see also Fig. 7). When Arg-138
is exchanged for lysine, Pmt1p-Pmt2p complexes (Fig. 6C,
compare lanes 1 and 4) as well as
O-mannosyltransferase activity (Fig. 7) are partially restored, indicating that a positive charged amino acid at that position is important for the establishment of functional Pmt1-Pmt2p complexes. Because this arginine residue is highly conserved between all PMT family members, we asked whether this residue also affects Pmt4p's homomeric interactions. We therefore replaced Arg-142 of
Pmt4pFLAG (the equivalent of Arg-138 in Pmt1p) with
glutamate using site-directed mutagenesis. The Pmt4p mutant protein
R142EFLAG was expressed and characterized in a yeast wild
type, a pmt4 and a pmt1pmt4 mutant strain.
SDS-PAGE and Western blotting of Triton extracts with polyclonal
anti-Pmt4p antibodies revealed that in wild-type yeast
Pmt4pFLAG and the mutant protein R142EFLAG show
an identical mass and are expressed at similar levels (data not shown).
However, Pmt4p-R142EFLAG failed to complement the
temperature-sensitivity of the pmt1pmt4 mutant, indicating
that Arg-142 is essential for Pmt4p activity in vivo (Fig.
7). In addition, coimmunoprecipitation experiments were performed on
Triton extracts of a wild-type strain coexpressing wild type Pmt4p and,
alternatively, Pmt4pFLAG, or Pmt4p-R142EFLAG
using monoclonal anti-FLAG antibodies. In contrast to
Pmt4pFLAG, which efficiently coimmunoprecipitates wild-type
Pmt4p, Pmt4p-R142EFLAG almost completely fails to
precipitate wild-type Pmt4p (Fig. 6D, compare lanes
1 and 2), consistent with a critical role for Arg-142
in Pmt4p-Pmt4p complex formation. Taken together, our data show that
similar principles underlie the formation of heteromeric complexes
between members of the PMT1 and PMT2 subfamilies and homomeric Pmt4p complexes.
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DISCUSSION |
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In yeast the PMT1 and PMT2 subfamilies but not the PMT4 subfamily
are highly redundant. In this study we demonstrate that the formation
of specific protein complexes is a common feature of PMT family members
in yeast. We found that, in general, members of the PMT1 subfamily
(Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily
(Pmt2p and Pmt3p). As schematically shown in Fig.
8A, Pmt1p-Pmt2p and
Pmt5p-Pmt3p are the predominant complexes formed between PMT1 and PMT2
subfamily members in wild-type S. cerevisiae cells. Under
certain conditions, however, Pmt1p can interact with Pmt3p, and Pmt5p
with Pmt2p, respectively. This can occur, for example, when one of the
principle partners is absent, as is the case in pmt mutant
strains. In contrast, the unique representative of the PMT4 subfamily
forms homomeric complexes (Fig. 8B). Interestingly, we
further uncovered that the same conserved protein domains influence
both heteromeric and homomeric Pmt-protein interactions.
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Heteromeric Protein Complexes between PMT1 and PMT2 Subfamily Members Might Have Evolved by Gene Duplication and Fulfill Similar Tasks in S. cerevisiae-- Within the PMT family in S. cerevisiae, Pmt1p is most closely related to Pmt5p (53% identity and 72% homology), and Pmt2p to Pmt3p (65% identity and 81% homology), respectively. In view of this high degree of conservation, the proteins Pmt1p and Pmt5p, as well as Pmt2p and Pmt3p might have evolved by gene duplication. This is supported by the fact that Pmt1p (YDL095w) and Pmt5p (YDL093w) are located directly next to each other on chromosome IV. Further, the report of Wolfe and Shields (42) suggests that Pmt2p (YAL023c; chr. I) and Pmt3p (YOR321w; chr. XV) constitute a protein pair that derives from an ancient duplication of the entire yeast genome. As a consequence of these gene duplication events, the ability to form specific protein complexes between individual members of the PMT1 and the PMT2 subfamily (Pmt1p-Pmt2p and Pmt5p-Pmt3p) evolved in S. cerevisiae. This connection also explains why Pmt1p-Pmt3p and Pmt5p-Pmt2p complexes can be formed, even though only under certain physiological conditions.
A series of observations suggest that Pmt1p-Pmt2p and Pmt5p-Pmt3p O-mannosyltransferase complexes act on the same protein substrates, albeit Pmt1p-Pmt2p complexes represent the predominant mannosyltransferase activity. Analyses of in vitro mannosyltransfer from Dol-P-Man to specific synthetic acceptor peptides have shown that the simultaneous deletion of PMT1 and PMT2 results in the loss of >80% of in vitro O-mannosyltransferase activity, when compared with wild-type yeast (1, 24). In addition, in vivo O-mannosylation of the same protein substrates is dramatically decreased in pmt1 and pmt2 mutant strains, such as the cell wall proteins Kre9p, chitinase (Cts1p), Bar1p, Ccw4p, and Ccw5p (14, 25). In contrast, in vitro and in vivo O-mannosylation is not obviously affected in pmt3 or pmt5 mutants (1, 14, 25). However, deletion of PMT3 in a pmt1pmt2 mutant further decreases in vitro mannosyltransfer from Dol-P-Man to a synthetic Pmt1p/Pmt2p-acceptor peptide by ~25%, when compared with the in vitro activity measured in pmt1pmt2 strains (1). In analyses of the O-mannosylation state of chitinase in pmt1pmt2 and pmt1pmt2pmt3 mutants, Gentzsch and Tanner (1) showed that in vivo chitinase is O-mannosylated by Pmt1p and Pmt2p as well as Pmt3p. Pmt3p, therefore, mannosylates the same proteins as Pmt1p and Pmt2p, but comes into operation mainly in the absence of Pmt1p and Pmt2p. The notion that Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes fulfill similar tasks in vivo is further supported by the fact that transcription of PMT1, PMT2, PMT3, and PMT5, but not PMT4 and PMT6 is enhanced in response to cell stress conditions that cause the accumulation of misfolded proteins in the ER (43). In summary, Pmt1p-Pmt2p and Pmt5p-Pmt3p are likely to O-mannosylate the same set of substrate proteins; yet, Pmt5p-Pmt3p complexes might, for example, exhibit lower substrate affinities and, therefore, play only a minor role in wild-type yeast cells. In the absence of Pmt1p and Pmt2p, however, Pmt5p-Pmt3p might compensate for O-mannosylation deficiency in pmt mutant strains. Similar functions might be assigned to the Pmt1p-Pmt3p and Pmt5p-Pmt2p complexes, which we could detect only in small amounts in wild-type strains (Figs. 2 and 3) or under specific physiological conditions such as in pmt mutants (Fig. 4). The observed increase of in vitro O-mannosyltransferase activity in pmt1pmt3 and pmt2pmt5 mutants (Table II) substantiates that Pmt1p-Pmt3p and Pmt5p-Pmt2p act as mannosyltransferases and feature substrate specificities similar to Pmt1p-Pmt2p. Our data suggest a compensatory cooperation between PMT1/PMT2 subfamily members, which might explain why in S. cerevisiae only the simultaneous deletion of several PMT subfamily members results in a substantial decrease in O-linked oligomannose chains and finally causes cell death (1). This possibility is supported by the fact that overexpression of PMT2 rescues the growth defect of a temperature-sensitive pmt1pmt4 mutant (data not shown). Because overexpression of PMT2 results in the formation of Pmt5p-Pmt2p complexes (Fig. 4), Pmt5p-Pmt2p might at least partially compensate the lack of Pmt1p-Pmt2p. Concordantly, Gentzsch et al. (21) showed that overexpression of PMT2 causes a slight increase of in vitro O-mannosyltransferase activity in wild-type yeast and in pmt1 deletion mutants. A compensatory cooperation between the redundant members of the PMT1/PMT2 subfamily in S. cerevisiae is also supported by the observation that in the fission yeast S. pombe where only one member of each PMT subfamily is present, the deletion of the single PMT2 subfamily member is lethal.2
The Third Member of the S. cerevisiae PMT2 Subfamily, Pmt6p, Interacts with None of the Other PMT Family Members-- In S. cerevisiae Pmt6p shares an overall sequence identity of 46% with Pmt2p and of 45% with Pmt3p. In the course of our analyses, BN-PAGE, coimmunoprecipitation, and chemical cross-linking experiments showed that Pmt6p interacts neither with Pmt1-5p nor with itself (data not shown), and therefore behaves differently from all other Pmtps in yeast. Nevertheless, BN-PAGE suggested that Pmt6p interacts with other proteins, although not with Pmtps (data not shown). To identify potential interacting partners, we analyzed whether selected components of the N-glycosylation machinery or the translocon are stably associated with Pmt6p. Again, no evidence was obtained that Wbp1p (40), Ost1p (44), Stt3p (45), or Sec61p (46) interact with Pmt6p (data not shown). Further studies are needed to elucidate the nature of these Pmt6p containing complexes.
Common Principles Underlie Heteromeric Pmt1p-Pmt2p and Homomeric Pmt4p-Pmt4p Interactions-- Considering Pmtps as antifungal targets it is noteworthy that in S. cerevisiae and in C. albicans only deletion of Pmt4p in combination with PMT1/PMT2 subfamily members causes lethality (1, 47). Therefore, to eliminate protein O-mannosylation both PMT1/PMT2 and PMT4 subfamily members must be inhibited. Pmt4p differs in several respects from PMT1/PMT2 subfamily members, such as substrate specificity and conserved signature sequence motifs (9, 14, 18). In addition, Pmt4p forms homomeric protein complexes as demonstrated in this study (Fig. 5). In view of these variances, it is of particular importance that common principles form the basis of the formation, structure and/or stability of PMT complexes in both PMT1/PMT2 and PMT4 subfamilies. Our mutational analyses showed that Pmt4p Arg-142, which is highly conserved between all PMT family members, is crucial for Pmt4p-Pmt4p complexes and enzyme activity (Figs. 6 and 7). Analogical, the corresponding mutation in Pmt1p affects Pmt1p-Pmt2p complexes and results in loss of mannosyltransferase activity (18). Thus, PMT complexes offer a point of attack to abolish protein O-mannosylation in fungi.
PMT Complexes Might Ensure Efficient O-Mannosylation--
There
are a number of reasons why protein O-mannosyltransferases
form homo- or heteromeric protein complexes. One is that these
complexes ensure an efficient O-glycosylation of a wide range of proteins. A common feature of O-glycosylated
proteins is that O-linked carbohydrate chains are clustered
in distinct serine/threonine rich regions (48). Such areas are thought
to adopt rod-like structures important for protein function. With a few
exceptions O-mannosylation occurs while proteins are
translocated in the lumen of the ER (49,
11).3 Thus, the clustering of
O-linked sugars requires high efficiency sugar transfer,
which might be provided by mannosyltransferase complexes. That
oligomerization enhances enzyme function has been proven for other
glycosyltransferases such as UDP-GlcNAc:dolichol-P GlcNAc-1-P
transferase (50) or the mannosyltransferase complexes M-Pol I and M-Pol
II (51-53). Furthermore, even though members of the PMT1 and PMT2
subfamily act on the same protein substrate (14, 25), they might
actually O-mannosylate different serine and threonine
residues within one and the same protein. Since to date no specific
consensus sequences are known that are required for
O-mannosylation this assumption remains to be verified.
However, this hypothesis is further supported by the fact that mutant
-factor precursor is O-mannosylated by Pmt2p but no other
PMT family member (11). To understand the functioning of PMT complexes
it will be important to learn more about their different substrate
specificities and what features of PMTs determine specificity.
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ACKNOWLEDGEMENTS |
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We thank C. Endres, M. Priesmeier-Gradl, and S. Lukas for excellent technical assistance and C. Frank, J. Klar, T. Seidl, and H. Wegele for providing assistance with the making of strains, plasmids, and anti-Pmtp antibodies, respectively. We are grateful to L. Lehle, J. Stolz, and U. Schermer for generously providing antibodies and plasmids, respectively. We thank W. Tanner for many helpful discussions and P. Orlean for the critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft (SFB521).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Lehrstuhl für
Zellbiologie und Pflanzenphysiologie, Universität Regensburg, 93040 Regensburg, Germany. Fax: 49-941-943-3352; E-mail:
sabine.strahl-bolsinger@biologie.uni-regensburg.de.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M212582200
2 T. Willer and S. Strahl, unpublished data.
3 I. Hagen and S. Strahl, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; PMT, protein O-mannosyltransferase; aa, amino acid; BN-PAGE, blue native polyacrylamide gel electrophoresis; Dol-P-Man, dolichyl phosphate-activated mannose; HA, hemagglutinin; OPA, o-phtaldialdehyde; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone.
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