From the Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, 93040 Regensburg, Germany
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ABSTRACT |
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The identification of the evolutionarily
conserved family of
dolichyl-phosphate-D-mannose:protein
O-mannosyltransferases (Pmts) revealed that protein O-mannosylation plays an essential
role in a number of physiologically important processes.
Strikingly, all members of the Pmt protein family share almost
identical hydropathy profiles; a central hydrophilic domain is flanked
by amino- and carboxyl-terminal sequences containing several putative
transmembrane helices. This pattern is of particular interest because
it diverges from structural models of all glycosyltransferases
characterized so far. Here, we examine the transmembrane topology of
Pmt1p, an integral membrane protein of the endoplasmic reticulum, from Saccharomyces cerevisiae. Structural predictions were
directly tested by site-directed mutagenesis of endogenous
N-glycosylation sites, by fusing a topology-sensitive
monitor protein domain to carboxyl-terminal truncated versions of the
Pmt1 protein and, in addition, by N-glycosylation scanning.
Based on our results we propose a seven-transmembrane helical model for
the yeast Pmt1p mannosyltransferase. The Pmt1p amino terminus faces the
cytoplasm, whereas the carboxyl terminus faces the lumen of the
endoplasmic reticulum. A large hydrophilic segment that is oriented
toward the lumen of the endoplasmic reticulum is flanked by five
amino-terminal and two carboxyl-terminal membrane spanning domains. We
could demonstrate that this central loop is essential for the function of Pmt1p.
Glycosylation is one of the most elaborate covalent protein
modifications known. The carbohydrate chains can be coupled to the
protein through either an N- or O-glycosidic
bond. Protein O-mannosylation, originally observed in fungi
(1), is initiated at the endoplasmic reticulum by protein
mannosyltransferases
(Pmts)1 that catalyze the
transfer of a mannosyl residue from dolichyl phosphate-activated
mannose (Dol-P-Man) to serine or threonine residues of nascent proteins
entering the secretory pathway; in the Golgi apparatus additional
sugars are added to the O-linked mannose with GDP-mannose
serving as carbohydrate donor (2, 3). Dol-P-Man-dependent
O-glycosylation of secreted proteins is a general feature of
yeasts and filamentous fungi (4).
The key enzyme of protein O-mannosylation, the
Dol-P-Man:protein O-mannosyltransferase Pmt1p, was purified
from Saccharomyces cerevisiae following the enzyme activity,
and the corresponding gene was cloned (5, 6). Pmt1p is an integral
membrane glycoprotein located at the ER (5, 7-9). Based on homology to
Pmt1p, a family of seven protein O-mannosyltransferases
(Pmt1p-Pmt7p) has been identified (10-13). Thus far, protein
O-mannosyltransferase activity has been demonstrated for
Pmt1p, Pmt2p, Pmt3p, Pmt4p, and Pmt6p (13, 14). The individual
mannosyltransferases recognize specific protein substrates that might
explain the presence of more than one transferase in S. cerevisiae (14). Moreover, Pmtp orthologues have been identified
from other yeasts (4), from the opportunistic fungal pathogen
Candida albicans (15), and from Drosophila
melanogaster (16) suggesting that protein
O-mannosylation may be common among eucaryotes.
The isolation of pmt mutants showed that protein
O-mannosylation plays a substantial role in a number of
physiologically important processes. In the yeast S. cerevisiae, protein O-mannosylation is an indispensable
modification for the maintenance of cell integrity (13). Deletion of
the PMT1 homologue in C. albicans results in
defects in morphogenesis, a significant loss of virulence, and reduced
adherence to host cells (15). In addition, mutations at the
Drosophila PMT1 orthologous locus, rotated
abdomen, alter muscle structures and the alignment of adult
cuticle (16). Despite the functional importance of the evolutionarily
conserved Pmtp mannosyltransferases, the initial steps of protein
O-mannosylation are still very poorly understood.
Pmtp family members are, on average, 50-55% homologous overall with
most variation occurring in the length and sequence of amino and
carboxyl termini. Most interestingly, all of the Pmts share a nearly
identical hydropathy profile, wherein an integral membrane protein with
a tripartite structure (amino- and carboxyl-terminal regions, each with
several putative transmembrane helices, and a central hydrophilic
segment) is predicted (6, 10, 11, 15, 16). Strikingly, this pattern
diverges from structural models of other ER glycosyltransferases as
well as from the common type II model of glycosyltransferases of the
Golgi apparatus.
In the present study we report the mapping of the membrane topology of
S. cerevisiae Pmt1p using site-directed mutagenesis, carboxyl-terminal reporter fusions, and N-glycosylation
scanning. These topology-sensitive monitors can distinguish between the lumen of the ER and the cytoplasm. We propose a structural model indicating that Pmt1p spans its cognate membrane seven times. In
addition, we demonstrate that a large luminally oriented hydrophilic loop is essential for Pmt1p function.
Yeast Strains
The S. cerevisiae strain STY50 (MATa,
his4-401, leu2-3, -112,
trp1-1, ura3-52, HOL1-1,
suc2::LEU2) was derived from the strain FC2a (17)
by disruption of the SUC2 gene by homologous recombination. For this purpose, FC2a was transformed with the plasmid pRR8.01 (kindly
provided by L. Lehle, University of Regensburg) digested with
HindIII. Yeast shuttle vectors YEp352 (2 µm,
URA3) (18), pR90 (PMT1R90, 2 µm,
URA3; see below) to pC731 (PMT1C731,
2 µm, URA3; see below) were transformed into the strain
STY50. The pmt1 deletion strain pmt1 Plasmid Constructions
Standard procedures were used for all DNA manipulations (21).
All cloning and transformations were carried out in Escherichia coli host DH5 PMT1-HIS4C Fusion Plasmids--
A 1.26-kilobase pair
AseI-HindIII fragment (bp Plasmid pSB52 (PMT1G355HA)--
A 111-bp
NotI fragment encoding three copies of the hemagglutinin
(HA) epitope was isolated from pAxl2 (22) and subcloned into pGEMEX-1
(Promega). Sequence analysis was used to identify clones with the
5'-sequence of the HA epitope following the XhoI site of the
vector. The HA epitope sequence (XhoI-SphI
fragment) was further subcloned from this construct (pSB50) into pG355
(XhoI, PflmI-digested), resulting in pSB52.
Plasmid pSB53 (PMT1)--
A carboxyl-terminal fragment of
PMT1 was amplified on genomic DNA using the primer pair
oligo Al4 (5-tcttgttatggttacagcgg-3') and oligo 133 (5'-tcactagcatgcggatccaccttcagcaaatg-3'). The PCR-fragment was digested
with PflmI and SphI and subcloned into pC731 (cut with
PflmI, SphI).
PMT1 Mutants--
Deletions or insertions of
N-glycosylation sites were attained by site-directed
mutagenesis using the QuickChangeTM Site-directed
Mutagenesis Kit from Stratagene. To create pSB57 (PMT1N743A) and pSB60
(PMT1N390A) the plasmid pSB53 was used as
template DNA. The primer pairs oligo 124 (5'-gaagagtacaaagcccaaaccttgactaaacgt-3') and oligo 125 (5'-cacgtttagtcaaggtttgggctttgtactcttc-3') were utilized to make pSB57,
oligo 122 (5'-acaacattccaagccctaaccgatggtaccaaggtc-3') and oligo 123 (5'-gaccttggtaccatcggttagggcttggaatgttgt-3') to make pSB60. pSB52
served as the template DNA to produce pSB62 (PMT1G355HA/loop 1)), pSB59
(PMT1G355HA/loop 4)), and pSB61
(PMT1G355HA/loop 6)). pSB62 was created
using the primer pair oligo 138 (5'-cctcctcttgcaaagaacttgtctgctggtaacgcatcgcttggtggg-3')/oligo 139 (5'-cccaccaagcgatgcgttaccagcagacaagttctttgcaagaggagg-3'), pSB59 the
primer pair oligo 117 (5'-gcaagcaaggacttgttagcattggtcgagttgtcagggtacat-3')/oligo 118 (5'-atgtaccctgccaactcgaccaatgctaacaagtccttgcttgc-3'), and pSB61 the
primer pair oligo 140 (5'-ggcgcaagcaacttttcgcctgaatttaactctacactaaagaac-3')/oligo 141 (5'-gttctttagtgtagagttaaattcaggcgaaaagttgcttgcgcc-3'). To create
pSB79 (PMT1 Computer Analyses
Structural predictions of Pmt1p were made using the programs
TMAP (23) and TMPRED. The latter uses an algorithm based on the
statistical analysis of TMbase (24). Furthermore, structural models
made by Martinsried Institute for Protein Sequences accession number
A47716 and SWISS-PROT accession number P33775, were used.
Analysis of the Pmt1-His4C Fusion Proteins
Growth on Histidinol--
The strain STY50 was transformed with
the plasmids pR90 to pC731. Transformants were selected for the
URA3-containing plasmids on SD plates supplemented with the amino acids
and bases required at 20-30 mg/liter, lacking uracil and containing
2% glucose. Ura+ transformants were streaked on
supplemented minimal medium lacking histidine but containing 6 mM histidinol. The plates were incubated at 30 °C for
3-5 days.
Immunoprecipitation from Whole-cell Extracts--
Yeast cells
were grown on SD medium to a concentration of 2.0 × 107 cells/ml. Cells (50 ml) were harvested and whole-cell
extracts prepared as described previously (25). 10 µl of
anti-invertase antibody (26) were added to 400 µl of whole-cell
extract and incubated for at least 3 h at 4 °C. Thereafter, 400 µl of lysis buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% sodium
deoxycholate, 2% Triton X-100, 0.1% SDS, 1 mM
phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.25 mM 1-chloro-3-tosylamido-7-amino-2-heptanone, 50 µg/ml
L-1-tosylamido-2-phenylethyl chloromethyl ketone, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) were added and the extracts dissected. 15 µl of bead volume protein A-Sepharose C-4B beads (Pharmacia) were added, and the incubation was
continued for 1 h at 4 °C. The immunoprecipitates were washed five times with 1.4 ml of lysis buffer and once with 1.4 ml of 50 mM potassium phosphate buffer, pH 5.5, 0.02% SDS, protease inhibitors as above. Subsequently, the precipitates were subjected to
endoglycosidase H digestion or mock treated.
Preparation of Crude Membranes
Yeast cells were grown on SD medium. At a concentration of
2.0 × 107 cells/ml, 20 ml of cells were harvested,
washed with 10 ml of 50 mM Tris-HCl, pH 7.5, 0.3 mM MgCl2, and resuspended in 100 µl of the
same buffer plus protease inhibitors (see above). An equal volume of
glass beads was added, and the cells were lysed by vortexing, for 1 min, four times (with 1-min intervals on ice). The bottom of the tube
was punctured and the lysate collected. Cell debris were removed by
centrifugation for 5 min at 3,000 rpm at 4 °C. Membranes were
collected from the supernatant by centrifugation for 30 min at 20,000 rpm at 4 °C and resuspended in 100 µl of 50 mM
Tris-HCl, pH 7.5, 7.5 mM MgCl2.
Isolation of Chitinase
Yeast cells were grown on SD medium to 2.0 × 107 cells/ml. Chitinase (Cts1p) was isolated from cell
walls as described in Gentzsch and Tanner (14).
Deglycosylation by Endoglycosidase H Digestion
Immunoprecipitates or 5 µl of crude membranes were suspended
in 25 µl of Endo H buffer (50 mM potassium phosphate
buffer pH 5.5, 0.02% SDS, 0.1 M 2-mercaptoethanol,
protease inhibitors as above) and digested with 1-5 units/µl of Endo
H for 1 h at 37 °C. Mock samples were incubated without Endo H. The reaction was stopped by adding 10 µl of 5× SDS sample buffer.
Western Blot Analyses
Proteins were fractionated on SDS-polyacrylamide gels and
transferred to nitrocellulose (27). Anti-Pmt1p and anti-invertase polyclonal antibodies were used at 1:1,000, anti-Cts1p polyclonal antibody at 1:2,500, and anti-HA monoclonal antibody (16B12; Babco) at
1:5,000 dilution. Protein-antibody complexes were visualized by
enhanced chemiluminescence using the Amersham Pharmacia Biotech ECL system.
In Vitro Dol-P-Man:Protein O-Mannosyltransferase Assay
5-30 µg of membrane protein were incubated in the in
vitro assay for Dol-P-Man:protein
O-mannosyltransferase, as described previously (5). The
pentapeptide acetyl-YATAV-NH2 was used at a final
concentration of 3.5 mM.
The Central Hydrophilic Loop and the Carboxyl-terminal End of Pmt1p
Are Facing the ER Lumen--
S. cerevisiae Pmt1p is a
protein of 817 amino acids with three potential
N-glycosylation sites as follows: two are located in the
central hydrophilic loop (aa Asn-390 and Asn-513) and one at the
carboxyl-terminal end (aa Asn-743) of the protein (Fig. 1). Treatment with endoglycosidase H
(Endo H) reduces the molecular mass of the protein from 92 to 84 kDa
(Fig. 2, lanes 2 and
3) (5). Considering the fact that Pmt1p resides in the ER
(7-9) where only core glycosylation takes place, this difference in molecular mass indicates that all three N-glycosylation
sequences (NX(S/T)) are glycosylated in vivo.
Since N-glycosylation is carried out exclusively on the
lumenal side of the ER, these data indicate that the
N-glycosylation sites are exposed to the ER lumen. To verify
this predicted orientation, Pmt1p mutant proteins were constructed
wherein the N-glycosylation sequons N390LT and N743QT were
destroyed individually by changing the asparagines to alanine. The
mutant mannosyltransferases were analyzed in the S. cerevisiae pmt1 deletion strain pmt1 Carboxyl-terminal Pmt1-His4C Reporter Fusions Reveal the Presence
of Seven Membrane Spanning Domains--
Resting upon the results of
the Pmt1pN390A/Pmt1pN743A mutant analyses we
used computer based algorithms (see "Experimental Procedures") to
propose three structural models of Pmt1p that featured 7, 10, or 11 transmembrane domains. To distinguish between the three models we used
fusion constructs of Pmt1p and a truncated version of the His4p protein
(His4C) as topology-sensitive reporters. His4C maintains histidinol
dehydrogenase activity and is translocated through the ER membrane when
fused to a signal sequence (28). Yeast his4 mutant strains
expressing a His4C fusion protein are able to grow on minimal medium
containing histidinol when the catalytic domain is present on the
cytoplasmic side of the ER membrane. In this case histidinol is
metabolized to histidine, resulting in a His+ phenotype.
When the catalytic domain is targeted to the ER lumen histidinol cannot
be converted to histidine, resulting in a his
We designed a series of fusion proteins consisting of carboxyl-terminal
truncated versions of Pmt1p and the His4C protein domain
(Pmt1R90 to Pmt1C731; Fig. 1 and Table
II) which allowed us to distinguish
between distinct numbers of transmembrane domains as well as their
orientation. In addition, the constructs contained a part of the yeast
invertase introducing an epitope for immunopurification. The fusion
constructs were transformed into a his4 mutant background
(STY50), and the transformants were tested for the ability to grow on
selective medium supplemented with histidinol. Furthermore, using a
polyclonal antibody directed against an unglycosylated form of
invertase (26), the fusion proteins were immunoprecipitated from
whole-cell extracts, treated with Endo H, and analyzed by Western
blot.
First we wanted to distinguish between an odd versus an even
number of membrane spanning domains. The number of transmembrane helices dictates the lumenal or cytoplasmic orientation of the amino
and carboxyl termini. The 10 transmembrane helical model requires that
both termini face the ER lumen. On the other hand, in case of a protein
with 7 or 11 transmembrane helices where the carboxyl terminus faces
the ER lumen, the amino terminus would face the cytoplasm.
To determine the orientation of the amino terminus the deletion
construct Pmt1R90 carrying the first putative transmembrane
domain (Fig. 1) was analyzed. As shown in Fig.
3, Pmt1R90 does not support
growth on histidinol indicating that His4C is facing the lumen of the
ER. This is validated by the fact that the fusion protein is highly
glycosylated in vivo (Fig. 4,
lanes 3 and 4). Pmt1R90 shows an
apparent molecular mass of 167 kDa which decreases after Endo H
treatment to 132 kDa. This is in agreement with the calculated mass of
the unglycosylated protein (Table II). These results indicate that the
amino terminus of this construct resides on the cytoplasmic side of the
ER membrane. A very minor fraction of Pmt1R90, which varied
in its abundance from experiment to experiment, is not glycosylated
(Fig. 4, lane 3). We presume that this fraction is either
oriented with the His4C domain in the cytoplasm or not translocated to
the ER at all, remaining misfolded in the cytoplasm. The latter
possibility would explain why this protein does not provide any growth
on histidinol (Fig. 3).
To confirm that the carboxyl terminus faces the ER lumen we used the
reporter fusion Pmt1C731, which contains all the potential
transmembrane domains. Pmt1C731 produces a
His
Next, we wished to discriminate between the 7 and the 11 transmembrane
helical model. In the fusion protein Pmt1R157 the His4C
domain is oriented toward the cytoplasm as indicated by the growth of
the transformants on medium supplemented with histidinol (Fig. 3).
Accordingly, the mobility of the protein on SDS-PAGE is not affected by
Endo H treatment showing that His4C is not glycosylated (Fig. 4,
lanes 5 and 6). Evidently, the catalytic His4C
domains of Pmt1R90 and Pmt1R157 are on
different sides of the ER membrane. The fusion Pmt1V175 is
facing the ER lumen (Fig. 3 and Fig. 4, lanes 7 and
8) and Pmt1L221 the cytoplasmic side of the
membrane (Fig. 3 and Fig. 4, lanes 9 and 10).
These data confirm that the predicted transmembrane domains TM I, TM
II, TM III, and TM IV (Fig. 1) are spanning the membrane in
vivo. Unexpectedly, the catalytic domain of the fusion Pmt1S263 is also facing the cytoplasm (Fig. 3 and Fig. 4,
lanes 11 and 12) implying that TM V does not
cross the membrane. The His4C domains of both fusion proteins
Pmt1P306 and Pmt1G355 are located in the ER
lumen (Fig. 3 and Fig. 4, lanes 13-16) indicating that TM
VI, but not TM VII (Fig. 1), traverses the membrane in vivo.
These data argue for the presence of five membrane spanning domains in
the amino-terminal half of Pmt1p.
How many transmembrane helices are present between the hydrophilic
middle part and the carboxyl terminus? To answer this question the
fusion proteins Pmt1P616 and Pmt1H655 were
analyzed. Pmt1P616 is oriented with the His4C catalytic
domain on the cytoplasmic side of the ER membrane (Fig. 3). Therefore,
TM VIII (Fig. 1) does span the membrane in vivo. A minor
increase in the mobility of the protein after Endo H treatment is due
to the removal of two N-linked carbohydrate chains at
positions Asn-390 and Asn-513 in the Pmt1p portion of the fusion (Fig.
4, lanes 17 and 18). A very similar result was
obtained for the fusion protein Pmt1H655 (Fig. 3 and Fig.
4, lanes 19 and 20) showing that TM IX does not
cross the membrane. These data, in combination with the prediction of
an odd number of transmembrane helices between the central hydrophilic
loop and the carboxyl-terminal end, demonstrate the presence of two
transmembrane helices in the carboxyl-terminal half of Pmt1p.
Glycosylation Scanning Mutagenesis Substantiates the Prediction of
Five Transmembrane Helices in the Amino-terminal Region of
Pmt1p--
Our results of the His4C fusion experiments favor the 7 transmembrane helical model with the helices TM I, TM II, TM III, TM
IV, and TM VI but not TM V and TM VII (Fig. 1) serving as membrane spanning domains in the amino-terminal half of the protein. On the
other hand, computer programs analyzing the Pmt1p hydropathy profile
(see "Experimental Procedures") predicted TM V to be a transmembrane helix. To verify that TM V does not span the membrane in vivo we used N-glycosylation scanning
mutagenesis. Since the high molecular weight of the native Pmt1p, its
own N-glycosylation, and its residence in the ER complicate
this kind of analysis, we circumvented these problems by constructing a
truncated version of Pmt1p, Pmt1G355HA (Fig.
5A), where the
carboxyl-terminal amino acids 356 to 817 are substituted with the
hemagglutinin (HA) epitope (29). This protein has a calculated mass of
45.3 kDa, no endogenous N-glycosylation sites, and is
immunologically detectable. On SDS-PAGE Pmt1G355HA migrates
at 43.5 kDa (Figs. 5B and 6C, lanes 1 and
2). In addition, we detected a second minor species with an
apparent mass of 42 kDa varying in its intensity in different
experiments. This species is assumed to be either a degradation product
or a modified version of the 43.5-kDa protein. The carboxyl terminus of
Pmt1G355HA is facing the ER lumen (data not shown) and thus
is consistent with the orientation of the central hydrophilic loop (aa
295-580) of the Pmt1p protein. Therefore, we concluded that this
protein reflects the transmembrane topology of the native Pmt1p
protein.
To confirm that the amino terminus faces the cytoplasm, we used
Pmt1G355HA and introduced two N-glycosylation
sequons into the loop region between the transmembrane helices TM I and
TM II by site-directed mutagenesis (Fig. 5A and Table
III). The N-glycosylation
sites were placed at least 12 amino acids away from adjacent membrane helices to ensure they could become N-glycosylated
(30). The resulting construct Pmt1G355HA/loop 1 was
expressed from a high copy 2-µm plasmid in the pmt1 mutant
strain pmt1
To answer the question as to whether TM V is used as a transmembrane
span in vivo, we independently introduced two
N-glycosylation consensus sequences in the loop regions
between TM IV and TM V (Pmt1G355HA/loop 4; Table III) and
between TM VI and TM VII (Pmt1G355HA/loop 6; Table III).
Considering the odd number of transmembrane helices predicted to form
between the amino terminus and the hydrophilic middle region, it may be
expected that either both TM V and TM VII serve as transmembrane spans
or neither of them. In the first case loop 4 and loop 6 would be
localized in the cytoplasm (Fig. 6A); in the second case loop 4 and loop 6 had to be on opposite sides of the membrane wherein loop 6 is facing the ER lumen (Fig. 6B). The mutant proteins
Pmt1G355HA/loop 4 and Pmt1G355HA/loop 6 were
expressed in the strain pmt1 The Central Hydrophilic Loop Is Crucial to Pmt1p
Function--
Summarizing, our data ascertain five transmembrane spans
between the amino terminus and a large central hydrophilic loop region which is facing the ER lumen and two membrane spanning domains between
the latter and the carboxyl terminus. The luminally oriented middle
loop (aa 295-580) is the largest hydrophilic segment in Pmt1p. Since
it is almost certain that the transfer of the mannose from Dol-P-Man to
proteins occurs in the ER lumen (31, 32), this loop might be essential
for Pmt1p function. To test this, we examined whether the large
hydrophilic loop is crucial for Pmt1p enzymatic activity.
We created a mutant version of Pmt1p (Pmt1
To test whether Pmt1 In this study, we present the first analysis of the transmembrane
topology of a Pmt-mannosyltransferase, an enzyme crucial to initiating
protein O-mannosylation at the ER. Our data provide strong
genetic and biochemical evidence for a seven-transmembrane helical
model, summarized in Fig. 8. The Pmt1p
amino terminus faces the cytoplasm, the carboxyl terminus the ER lumen.
A large hydrophilic region, located in the ER lumen, is separated from the amino terminus by five and from the carboxyl terminus by two membrane spanning domains. By using deletion mutagenesis we show that
the ER luminally oriented central loop is crucial for
mannosyltransferase activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(MAT
,
his3-
200, leu2-3, -112, lys2-801,
trp1-
901, ura3-52, suc2-
9,
pmt1::HIS3) (13) was transformed with the shuttle
vectors YEp352, pSB53 (PMT1, 2 µm, URA3; see
below), pSB57 (PMT1N743A, 2 µm,
URA3; see below), pSB60 (PMT1N390A, 2 µm, URA3; see below), pSB52
(PMT1G355HA, 2 µm, URA3; see
below), pSB62 (PMT1G355HA/loop 1), 2 µm, URA3; see below), pSB59
(PMT1G355HA/loop 4), 2 µm,
URA3; see below), pSB61 (PMT1G355HA/loop
6), 2 µm, URA3; see below), pSB73
(PMT1, 2 µm, URA3; see below) or pSB79
(PMT1
304-531, 2 µm, URA3; see
below). All yeast transformations were performed following the method
of Gietz et al. (19). SUC2 gene disruption was
confirmed measuring invertase activity (20).
. PCR fragments were routinely checked by sequence analysis.
343 to +914 of
PMT1) was subcloned from pDM3 (6) into pBluescript KS+
(Stratagene) digested with HindIII and SmaI
resulting in the plasmid pK1A. The PMT1 coding region from
bp +889 to +1065 was amplified by PCR with the primer pairs oligo Al1
(5'-actttggacggggatggc-3') and oligo Al2
(5'-gactcgctcgagaccagctggataattgtg-3'; XhoI site is underlined). The PCR fragment was subcloned as a
HindIII-XhoI fragment into pK1A
(HindIII-XhoI). From the resulting plasmid, pK1,
the ScaI-XhoI fragment was subcloned into pA
7
(digested SacI, XhoI) carrying the
SUC2-HIS4C fusion construct (17) resulting in plasmid pG355.
pR90 was constructed by amplifying PMT1 from bp
137 to
+270 with oligo Al3 (5'-tgtcgaagaagagtttggcg-3') and oligo Al5
(5'-ttaccgctcgagcctaatgtattgcgaggc-3'), digesting the fragment with BglII-XhoI, and subcloning it
into pK1 (digested BglII and XhoI). The
SacI-XhoI fragment of the resulting plasmid was
then subcloned into pA
7. The construction of pR157 was analogous to
that of pR90 using oligos Al3 and Al6
(5'-ttaccgctcgagacgtaaagtcatgtacatc-3'). For pV175 oligo
Al3 and oligo 105 (5'-acgtacgactcgagaacggcaaagcagatagcgctc-3'), for pL221
oligos Al3 and Al10 (5'-ttaccgctcgagaagcaaggacttgtaagc-3') were used to amplify PMT1 from bp
137 to +525 and bp
137
to +659, respectively. The PCR fragments were digested with
Bsh1365I and XhoI and subcloned into pR157 (cut
with Bsh1365I and XhoI). For pS263 to pC731 the
following regions of PMT1 were amplified by PCR: pS263, bp
+550 to +789, oligos Al9 (5'-cgttacattctgttggacgc-3') and Al11
(5'-ttaccgctcgagggaagacttagtcaaatcc-3'); pF306, bp +550 to
+918, oligos Al9 and Al12
(5'-cattagctcgagaaagaagcttgcgccatcc-3'); pP616, bp +550 to
+1848, oligos Al9 and Al7
(5'-ttaccgctcgagtggtttacctaactgcc-3'); pH655, bp +550 to
+1965, oligo Al9 and oligo101
(5'-ttaccgctcgaggtgatgcaaaaacatttgacgttg-3'); pC731, bp
+550 to +2193, oligos Al9 and Al8
(5'-ttaccgctcgagacaattgtagtcccaacc-3'). The PCR fragments
were digested with PstI and XhoI and subcloned into pL221 cut with the same.
304-531), the plasmid pSB53 was
digested with SphI, NarI, and religated. The
resulting plasmid (pSB73) was cut with HindIII and
religated, thereby deleting the PMT1 sequence coding for aa
304 to aa 531.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Pmt1pN390A and
Pmt1pN743A are functionally active as demonstrated by their
in vitro mannosyltransferase activity (Table
I). Western blot analysis showed that the
molecular mass of both mutant proteins decreases from 92 to 89 kDa
compared with wild type Pmt1p (Fig. 2, lanes 3-5). This is
consistent with the loss of one N-linked sugar chain,
demonstrating that the sequons N390LT and N743QT are
N-glycosylated in vivo. From these results we
conclude that the central hydrophilic loop (aa 295-580) and the
carboxyl-terminal end (aa 720-817) of Pmt1p are facing the ER
lumen.
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Fig. 1.
Carboxyl-terminal Pmt1-His4C reporter fusion
constructs. The hydropathy profile of Pmt1p using a window of 17 amino acids is shown (60). Solid diamonds indicate
N-glycosylation sites. Potential transmembrane spanning
domains are marked by roman numerals. The terminal amino
acids of the Pmt1 portion in the individual Pmt1-His4C fusion proteins
are shown.
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Fig. 2.
Analysis of endogenous
N-glycosylation mutants. Crude membranes were
isolated from the yeast strains pmt1 /YEp352 (pmt1
; lane
1), pmt1
/pSB53 (PMT1; lanes 2 and
3), pmt1
/pSB60 (PMT1N390A;
lane 4), and pmt1
/pSB57
(PMT1N743A, lane 5) and treated with
Endo H as indicated. Proteins (25 µg) were resolved on 8%
SDS-polyacrylamide gels and analyzed by Western blot using an
anti-Pmt1p antibody.
Effects of mutations in N-glycosylation sequons on
O-mannosyltransferase activity
. Crude membranes were isolated
as described under "Experimental Procedures." 5-30 µg of protein
were incubated in the in vitro mannosyltransferase assay
following the transfer of [14C]mannose from Dol-P-Man to the
pentapeptide Ac-YATAV-NH2 (5). Average values of a typical
experiment are shown.
phenotype.
In addition, the protein becomes extensively glycosylated due to the
presence of several N-glycosylation sites.
Pmt1-His4C fusion proteins
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Fig. 3.
Growth phenotypes of Pmt1-His4C fusions.
The yeast strain STY50 was transformed with the plasmid YEp352 or
plasmids coding for the Pmt1-His4C fusions Pmt1R90 to
Pmt1C731 (Arg-90 to Cys-731). Transformants were streaked
on selective media supplemented with histidine (left panels)
or histidinol (right panels) and incubated for 3-5 days at
30 °C.
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Fig. 4.
Analysis of the N-
glycosylation state of the Pmt1-His4C fusion proteins. The
Pmt1-His4C fusion proteins Pmt1R90 to Pmt1C731
were immunoprecipitated from whole-cell extracts made from STY50
transformed with the plasmid YEp352 (lanes 1 and
2) or plasmids pR90 to pC731 (lanes 3-22) using
a polyclonal anti-invertase antibody. Immunoprecipitates were treated
with Endo H as indicated and separated on 7% SDS-polyacrylamide gels.
Western analysis was performed using an anti-invertase antibody.
phenotype (Fig. 3) and is extensively glycosylated
(Fig. 4, lanes 21 and 22) indicating that the
His4C domain is oriented toward the ER lumen. Since the amino and
carboxyl termini are located on different sides of the ER membrane we
exclude the 10 transmembrane helical model. Furthermore,
Pmt1G355 shows the same phenotype as Pmt1C731
confirming the lumenal orientation of the hydrophilic central part of
Pmt1p (Fig. 3 and Fig. 4, lanes 15 and 16). Taken
together, these results indicate the presence of 7 or 11 transmembrane
domains with an odd number of helices between the amino terminus and
the middle loop and an even number between the latter and the carboxyl terminus of Pmt1p.
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Fig. 5.
Transmembrane topology of
Pmt1355HA. A, predicted transmembrane
topology of Pmt1355HA deduced from the Pmt1-His4C fusion
experiments. Arrows indicate the N-glycosylation
sites inserted in the construct Pmt1355HA/loop 1 (see also
Table III). Roman numerals mark the putative transmembrane
spanning domains. HA, hemagglutinin epitope. B,
crude membranes (25 µg of protein), isolated from the yeast strains
pmt1 /pSB52 (PMT1355HA; lanes 1 and
2) and pmt1
/pSB62 (PMT355HA/loop
1; lanes 3 and 4), were resolved on 10%
SDS-polyacrylamide gels after incubation with Endo H. Western analysis
was performed using a monoclonal anti-HA antibody.
. A crude membrane fraction was isolated and treated with
Endo H to verify N-glycosylation. The proteins were then
analyzed by Western blot using a monoclonal antibody directed against
the HA epitope. Our data show that the loop region between TM I and TM
II is N-glycosylated in vivo. Core-glycosylated
species of Pmt1G355HA/loop 1 were detected in addition to
the unglycosylated protein (Fig. 5B, lanes 3 and
4). It is likely that this partial glycosylation is due to
varied numbers of N-glycosylation sites being used. Similar
effects were observed when Pmt1p was expressed from a high copy 2-µm
plasmid.2 From these results
we conclude that the loop region between TM I and TM II is located in
the ER lumen and, consequently, that the amino terminus of
Pmt1G355HA is oriented toward the cytoplasm, confirming the
results obtained by His4C reporter fusions.
N-glycosylation sequons introduced in Pmt1G355HA
. To examine the state of glycosylation
crude membranes were isolated, treated with Endo H, and analyzed by
Western blot using a monoclonal anti-HA antibody. Fig. 6C
shows that Pmt1G355HA/loop 6 (lanes 5 and
6) but not Pmt1G355HA/loop 4 (lanes 3 and 4) is glycosylated in vivo providing further evidence that TM V does not serve as a membrane spanning helix. These
results demonstrate the presence of only five transmembrane spanning
domains in the amino-terminal half of Pmt1p.
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Fig. 6.
Analysis of amino-terminal transmembrane
helices. A and B, possible arrangements of
the transmembrane helices TM IV to TM VII. Putative membrane spans are
marked in roman numerals. L4, loop 4; L6, loop 6. C, Western analysis of crude membranes isolated from the
yeast strains pmt1 /pSB52 (PMT1355HA;
lanes 1 and 2), pmt1
/pSB59
(PMT1355HA/loop 4; lanes 3 and
4), and pmt1
/pSB61
(PMT1355HA/loop 6; lanes 5 and
6) was performed using a monoclonal anti-HA antibody.
Membranes (25 µg of protein) were treated with Endo H as
indicated.
304-531; see
"Experimental Procedures") that lacks the amino acid residues
304-531 including the N-glycosylation sequons Asn-390 and
Asn-513 (Fig. 1). Pmt1
304-531 was analyzed in a
pmt1 mutant background. On SDS-PAGE
Pmt1
304-531 shows an apparent mass of 64 kDa (Fig.
7A, lane 4), being similar to
the predicted size of 66.9 kDa. Endo H treatment reduces the mass to 62 kDa (Fig. 7A, lane 5). This decrease in mass of 2 kDa indicates that the only N-glycosylation site (Asn-743; see
Fig. 1) present in Pmt1
304-531 bears one
N-linked core carbohydrate chain. From these results we
conclude that (i) Pmt1
304-531 resides in the ER
membrane and (ii) Pmt1
304-531 mirrors the membrane
topology of native Pmt1p.
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Fig. 7.
The luminally oriented hydrophilic loop is
crucial for Pmt1p function. A, crude membranes (25 µg
of protein) isolated from the yeast strains pmt1 /YEp352
(pmt1
; lane 1), pmt1
/pSB73 (PMT1;
lanes 2 and 3), and pmt1
/pSB79
(PMT1
304-531; lanes 4 and
5) were resolved on a 10% SDS-polyacrylamide gel and
analyzed by Western blot using an anti-Pmt1p antibody. Membranes were
treated with Endo H as indicated. B, chitinase (Cts1p) was
isolated from the yeast strains pmt1
/YEp352 (pmt1
;
lanes 1 and 4), pmt1
/pSB73 (PMT1;
lane 2), and pmt1
/pSB79
(PMT1
304-531; lanes 3 and
5) and analyzed by Western blot as described by Gentzsch and
Tanner (14).
304-531 still has
mannosyltransferase activity, we analyzed the in vivo
glycosylation status of the highly O-mannosylated protein
chitinase (Cts1p; see Ref. 33) in a yeast pmt1 mutant expressing Pmt1
304-531. Confirming previous results
(14) we found that in the pmt1 deletion strain Cts1p is less
glycosylated as compared with a strain where Pmt1p is present (Fig.
7B, lanes 1, 2, and 4). The mutant protein
Pmt1
304-531 does not repeal the underglycosylation of
Cts1p (Fig. 7B, lane 3). Furthermore,
Pmt1
304-531 did not show significant in
vitro mannosyltransferase activity (data not shown). Since the
amounts of Pmt1
304-531 and native Pmt1p protein are
very similar in the strain pmt1
(Fig. 7A, lanes 2 and
4), these data definitively prove that the luminally
oriented hydrophilic loop is essential for Pmt1p function.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Predicted seven-transmembrane helical
structure of yeast Pmt1p. The amino and carboxyl termini face the
cytoplasm and the ER lumen, respectively. The central hydrophilic loop
is oriented toward the lumen of the ER and is flanked by five
amino-terminal and two carboxyl-terminal membrane spanning domains.
N-glycosylation sites are marked with solid
diamonds.
The successful use of heterologous protein fusions as topology-sensitive monitors (17, 34) encouraged us to use His4C as a reporter of the topological location which discrete portions of Pmt1p acquire in the membrane. The results obtained with Pmt1-His4C fusions are supported by several other lines of evidence as follows. (i) N-glycosylation scanning demonstrates that TM I integrates into the membrane with its carboxyl-terminal region reaching into the ER lumen. As a consequence the Pmt1p amino terminus is cytoplasmic. In agreement with these data TM I shows the features of a "type II signal anchor sequence" (reviewed in Ref. 35); TM I (aa 51-70) is amino-terminally flanked by three positive (Arg-42, Lys-48, and Lys-50) and carboxyl-terminally by three negative (Asp-72, Asp-77, and Glu-78) charges. (ii) Mutation of the endogenous Pmt1p N-glycosylation sequons N390LT and N743QT demonstrates that the central hydrophilic loop and the carboxyl terminus of Pmt1p are oriented toward the ER lumen. Earlier results resting upon Endo H digestion of Pmt1p isolated from yeast and heterologous expression of Pmt1p in E. coli already suggested that all three N-glycosylation sites (Asn-390, Asn-513, and Asn-743) are used in the yeast Pmt1p protein (5, 6). (iii) Our N-glycosylation scanning data corroborates the prediction that hydrophobic helix TM V does not span the membrane. TM V (aa 235-256) is flanked on both sides by positively charged loops and thus might adopt a "leave-one-out" topology as observed by Gafvelin and von Heijne (36) for E. coli inner membrane proteins. (iv) The majority of the loop regions between the seven transmembrane helices follow the "positive-inside rule" which states that positively charged residues are often found flanking hydrophobic transmembrane segments on the cytosolic side of the membrane (reviewed in Ref. 37).
The complex organization of yeast Pmt1p contrasts the structure of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases that initiate mucin-type O-glycosylation at the Golgi apparatus of higher eucaryotes (38). The GalNAc-transferases show small cytosolic amino-terminal domains, single transmembrane segments, lumenal stem regions, and large carboxyl-terminal lumenal domains responsible for catalysis. This type II model structure is common to glycosyltransferases of the Golgi apparatus, for example the yeast mannosyltransferases Mnt1/Kre2p (39, 40) or Mnn1 (41).
ER resident glycosyltransferases showing multiple putative transmembrane helices were found in S. cerevisiae as well as in higher eucaryotes. These include the glycosyltransferases involved in the synthesis of the lipid oligosaccharide precursor for N-glycans (3, 42) and the glycosylphosphatidylinositol (GPI) anchor (3, 43). The topological organization of these transferases is not well characterized, but for the ones investigated so far the putative transmembrane topology does not resemble the structure of Pmt1p. A scarce example where the structure-function relationship has been examined in detail is the hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT) (44, 45). This enzyme initiates N-linked glycosylation by catalyzing the synthesis of GlcNAc-P-P-dolichol. Lehrman and co-workers (42, 46) demonstrated that GPT is a multimeric enzyme with multiple, most likely 10 transmembrane spans. The largest hydrophilic segment, located between TM 9 and TM 10, is facing the cytoplasm. This loop region most likely bears the catalytic site consistent with the fact that GlcNAc-P-P-dolichol is synthesized on the cytoplasmic side of the ER. GPT seems to be highly conserved between higher eucaryotes and yeast since the human GlcNAc-1-P transferase complements a S. cerevisiae alg7 (asparagine linked glycosylation) mutant defective in GPT activity (47). Recently, several ER-located glycosyltransferases have been identified which, like Pmt1p, use dolichol phosphate-activated sugars as donor substrates. These include Alg3p (48), Alg6p (49), Alg8p (50), Alg9p (51), and Alg10p (52) from S. cerevisiae which participate in the assembly of the dolichol pyrophosphate-linked oligosaccharide at the lumenal side of the ER, the human PIG-B protein (53) required for GPI anchor synthesis, and its functional homologue Gpi10p from yeast (54). Computer analyses predict the presence of several transmembrane domains in the Dol-P-sugar-utilizing transferases, but only in the case of PIG-B has the membrane topology been investigated. Takahashi et al. (53) provided evidence that despite its hydrophobic nature PIG-B shows the topological structure of a type II membrane protein. PIG-B consists of a short amino-terminal cytoplasmic segment, a transmembrane domain, and a large carboxyl-terminal region facing the ER lumen. The discrepancy between the putative and experimentally determined structure of PIG-B demonstrates how cautious computer-based analyses should be interpreted and how important it is to obtain direct structural information.
Our analyses elucidate an elaborate structure for yeast Pmt1p with seven transmembrane domains and a number of loop regions. The large hydrophilic central loop (aa 295-580) is essential for Pmt1p activity, suggesting that the catalytic site is facing the ER lumen. This is in good agreement with previous data showing that Dol-P-Man is used as donor on the lumenal side of the ER membrane for the mannosylation of the N-glycan precursor intermediates, for the synthesis of GPI anchors as well as for protein O-mannosylation (55). Like for Pmt1p the catalytic domain of PIG-B is also facing the ER lumen since deletion of the cytoplasmic domain does not abolish enzymatic activity (53).
Since protein O-mannosylation is an essential modification in yeast (13), Pmt1p may be subject to stringent regulation. Thus, it is possible that some regions are involved in catalysis, whereas others could interact with various proteins or regulators. This assumption is sustained by the observation that Pmt1p interacts with Pmt2p in vivo, and the formation of this complex is required for maximum transferase activity (56). Interactions with different regulators are also conceivable and even suggested by the finding that Pmt proteins possess three highly conserved phosphorylation sites for protein kinase C (SX(R/K) and TX(R/K)) and pmt mutants display phenotypes similar to those observed in protein kinase C mutants (13, 57). In addition, S. cerevisiae and C. albicans Pmt1p can be activated by phospholipids in vitro (58).3 That Pmt O-mannosyltransferases are the subject of various regulation mechanisms is emphasized by the fact that yeast PMT1-PMT6 are also transcriptionally regulated during the cell cycle3 and during diauxic shift from fermentation to respiration (59).
Future work will be necessary to learn more about the mechanism
how Pmts transfer mannose from Dol-P-Man to specific acceptor proteins
at the endoplasmic reticulum. This study provides the basis for the
identification and characterization of structural and functional
important domains of Pmt O-mannosyltransferases.
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ACKNOWLEDGEMENTS |
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We are grateful to L. Lehle and M. Gentzsch for the gift of plasmids and to L. Lehle for generously providing antiserum to yeast invertase and Endo H. We thank W. Tanner, M. Gentzsch, V. Mrsa, and L. Lehle for many helpful discussions and, especially, M. Büttner, R. Mann, and W. Tanner for their critical comments on the manuscript. We also thank A. Benner for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft Grant 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: 49-0941-943-3352; E-mail:
sabine.strahlbolsinger{at}biologie.uni-regensburg.de.
2 M. Gentzsch, personal communication.
3 S. Strahl-Bolsinger, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: Pmt, protein O-mannosyltransferase; aa, amino acid; Dol-P-Man, dolichyl-phosphate-D-mannose; Endo H, endoglycosidase H; ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol; GPT, GlcNAc-1-P-transferase; His4C, histidinol dehydrogenase protein domain; TM, transmembrane domain; HA, hemagglutinin epitope; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); oligo, oligonucleotide(s); PCR, polymerase chain reaction.
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REFERENCES |
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