From the Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794-5215
Received for publication, October 5, 2000, and in revised form, November 3, 2000
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ABSTRACT |
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Glycoproteins and lipids in the Golgi complex are
modified by the addition of sugars. In the yeast Saccharomyces
cerevisiae, these terminal Golgi carbohydrate modifications
primarily involve mannose additions that utilize GDP-mannose as the
substrate. The transport of GDP-mannose from its site of synthesis in
the cytosol into the lumen of the Golgi is mediated by the
VRG4 gene product, a nucleotide sugar transporter that is a
member of a large family of related membrane proteins. Loss of
VRG4 function leads to lethality, but several viable
vrg4 mutants were isolated whose GDP-mannose transport
activity was reduced but not obliterated. Mutations in these alleles
mapped to a region of the Vrg4 protein that is highly conserved among
other GDP-mannose transporters but not other types of nucleotide sugar
transporters. Here, we present evidence that suggest an involvement of
this region of the protein in binding GDP-mannose. Most of the
mutations that were introduced within this conserved domain, spanning
amino acids 280-291 of Vrg4p, lead to lethality, and none interfere
with Vrg4 protein stability, localization, or dimer formation. The null
phenotype of these mutant vrg4 alleles can be complemented
by their overexpression. Vesicles prepared from vrg4 mutant
strains were reduced in luminal GDP-mannose transport activity, but
this effect could be suppressed by increasing the concentration of
GDP-mannose in vitro. Thus, either an increased substrate
concentration, in vitro, or an increased Vrg4 protein
concentration, in vivo, can suppress these vrg4
mutant phenotypes. Vrg4 proteins with alterations in this region were reduced in binding to guanosine 5'-[ The terminal carbohydrate modifications of proteins and lipids in
the Golgi complex are essential for life and play a variety of
important biological roles that serve to regulate cell surface properties. The substrates utilized by the luminal Golgi
glycosyltransferases are nucleotide sugars, whose site of synthesis is
the cytosol. Delivery of these molecules into the Golgi is mediated by
membrane-bound nucleotide sugar transporters
(NSTs).1 A large and growing
number of related membrane proteins in this family have been identified
in eukaryotes. NSTs differ from one another in their substrate
specificity and also in their cellular locations. Mammalian
glycoconjugates are modified by many different types of sugars,
requiring a correspondingly large number of different NSTs that are
localized in the ER, Golgi, and even the nucleus (see Refs. 1 and 2 for
reviews). Plants and lower eukaryotes also require nucleotide sugar
uptake into compartments of the secretory pathway for the biosynthesis
of certain extracellular polysaccharides that are structural components
of their cell wall but whose synthesis, at least in part, occurs
intracellularly. In plants, both hemicellulose and pectin are
synthesized in the Golgi (3, 4), and NST activities required for their
synthesis, including UDP-glucose and GDP-fucose transporters have been
characterized (5-7). Regulation of NST activity has been hypothesized
to be an important mechanism to regulate glycosylation, through the provision of substrate. This idea is supported by the severe
glycosylation phenotypes caused by mutations in various NST genes
(8-12).
The molecular details of nucleotide sugar recognition by the NSTs
remain unresolved. Early studies established that NSTs recognize both
the nucleotide and sugar portions of nucleotide sugars, although the
primary determinant for specificity resides in the nucleotide (13).
Although it was believed that each NST recognizes only one type of
nucleotide sugar, recent studies of the Leishmania GDP-mannose transporter suggest that this notion may apply to only a
subclass of NSTs. The Leishmania GDP-mannose transporter, whose physiological substrate is GDP-mannose, is capable of
transporting GDP-mannose, GDP-fucose, and GDP-arabinose in
vitro (14). Functional analyses of several NSTs, including the
yeast GDP-mannose transporter, the human UDP-galactose transporter, and
the murine and human CMP-sialic acid transporters identified domains
required for protein stability, folding, and transport (12, 15-17).
However, virtually nothing is known about what contributes to the
substrate specificity that distinguishes each of the NSTs.
In the yeast, S. cerevisiae, proteins and lipids are
modified in the Golgi primarily by the addition of mannose, using
GDP-mannose as the substrate. The VRG4 gene encodes the
yeast GDP-mannose transporter (18). As expected for the sole provider
of the substrate required for protein glycosylation in the Golgi,
VRG4 is an essential gene. Several viable mutants of the
yeast GDP-mannose transporter have been isolated that exhibit severe
glycosylation defects (17, 19, 20). Here we identify, by genetic and
biochemical analysis of the mutant proteins encoded by vrg4
alleles, a motif that is critically important for glycosylation and
nucleotide sugar transport but which, when mutated, does not interfere
with Vrg4 protein stability, Golgi localization, or multimerization
properties. This motif is highly conserved in GDP-mannose transporters
but not in other types of nucleotide sugar transporters, implying a
role in binding to nucleotide sugar or nucleoside monophosphate. Mutational analysis of this region demonstrated that an alteration of
most of the nonvariant amino acids in this region are required for
viability, but this phenotype can be complemented by overexpression of
these vrg4 alleles as a consequence of producing higher
levels of mutant Vrg4 protein in vivo. Biochemical analyses
of these mutant Vrg4 proteins demonstrated that their reduced transport activity could also be overcome by increasing substrate concentration. These mutant proteins also display a reduced affinity toward the photo
probe, [ Yeast Strains and Media--
Standard yeast media and genetic
techniques were used (21). Hygromycin B sensitivity was tested on yeast
extract/peptone/adenine sulfate/dextrose plates (YPAD) supplemented
with 30-50 µg/ml hygromycin B (Roche Molecular Biochemicals) as
described (19). NDY5 (MAT Cloning and DNA Sequence Analysis of the vrg4-2 Allele--
A
1.35-kb fragment containing the vrg4-2 open
reading frame and 237 base pairs of 5'-flanking and 72 base pairs of
3'-flanking sequences, was amplified by PCR using LA Taq
thermophilic DNA polymerase (TaKaRa Shuzo, Japan) from genomic DNA
isolated from the vrg4-2 strain, NDY5 (10). These
PCR products were directly cloned into the pCRII-TA cloning vector
(InVitrogen) to generate pCRIIvrg4-2. Plasmids from three independent
clones were isolated, and the sequence from each of these was compared
with the VRG4 gene isolated by PCR from the isogenic
parental strain and cloned in the same way to exclude PCR-derived
mutations. DNA sequencing was performed as described (22) using the
Thermo Sequenase cycle sequencing kit (Amersham Pharmacia Biotech),
using an automated LI-COR 4000L DNA sequencer.
Construction of Plasmids--
Standard molecular biology
techniques were used for all plasmid constructions (23).
To construct a series of equivalent expression plasmids containing
either the VRG4 or vrg4-2 allele that
differ only in the A286D mutation, at nucleotide position 857, a
251-base pair HpaI/MfeI fragment from
pCRIIvrg4-2, containing this point mutation, was used to replace the
same region in the wild type VRG4 gene.
The plasmid, pRSvrg4-A286D was made by replacing this
HpaI/MfeI fragment in pRHL (10), which contains
the VRG4 gene under its own promoter in pRS316, a
CEN6/URA3 vector (24). To introduce a C-terminal HA tag, the
3'-end of vrg4-A286D gene, lacking the point mutation, was
substituted with a 0.5-kb MfeI/SacI fragment from
SK
YEpGAPvrg4-A286D-HA3 was made by replacing the 251-base
pair HpaI/MfeI fragment from pCRIIvrg4-2,
containing the A286D mutation, in YEpGAPVRG4-HA3 (16). This
plasmid encodes HA-tagged vrg4-A286D under the control of
the glyceraldehyde-3-phosphate dehydrogenase (TDH3) promoter
in the 2µ/URA3 vector YEpGAP (25).
A series of plasmids containing mutant vrg4 alleles with a
single missense mutations that affect amino acids within the consensus motif (spanning amino acids 280-291) were created as follows. An
EcoRI/SalI fragment containing the entire wild
type VRG4-HA3 gene was subcloned into pBluescript
SK
To express these mutant vrg4 alleles at physiological levels
from the VRG4 promoter in a CEN-containing
vector, the HpaI/XbaI fragment in
pRHL-HA3 (18) was replaced with a 0.5-kb
HpaI/XbaI fragment from each of the
pSK
To express these mutant vrg4 alleles in yeast at high copy,
under the control of the TDH3 promoter in a 2µ vector,
EcoRI/XhoI fragments containing the entire
mutated vrg4-HA gene from each of these plasmids were
ligated into YEpGAP. This generated the series of mutant HA-tagged
vrg4 alleles, described above, in YEpGAP.
To construct integration plasmids, an EcoRI/SalI
fragment from pSK Coimmunoprecipitation, Western Immunoblotting, and
Immunofluorescence--
Exponentially growing yeast cells
(A600 = 1-3) were harvested and converted to
spheroplasts with lyticase, as described (27). Spheroplasts from 3-4
OD units of cells were resuspended in 400 µl of ice cold lysis buffer
(150 mM NaCl, 10 mM HEPES-KOH (pH 7.5), 5 mM MgCl2, 1 mM phenylmethylsulfonyl
fluoride) containing either 1% digitonin or 1% Triton X-100 to
solubilize membrane proteins and centrifuged for 5 min at 4 °C at
14,000 × g to remove debris. These detergent
extracts were used as the source for both Western blot analyses,
performed as described (16), and for the coimmunoprecipitation assays,
described below.
The HA-tagged or myc-tagged proteins were immunoprecipitated by
incubating 400 µl of a detergent extract with 200 µl of a hybridoma
cell culture supernatants containing the 12CA5 monoclonal anti-HA
antibody and 25 µl of protein A-Sepharose (Amersham Pharmacia Biotech) at room temperature for 2 h and processed for 10%
SDS-PAGE as described (16). Immunoblotting was performed with anti-myc A-14 polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA). Secondary anti-rabbit antibodies conjugated to horseradish peroxidase (Amersham Pharmacia Biotech) were used at a 1:3000 dilution
and detected by chemiluminescence (ECL, Amersham) followed by autoradiography.
Indirect immunofluorescence of yeast cells expressing Vrg4-HAp or
HA-tagged Vrg4 mutant proteins and data analyses were as described
(18).
[ Subcellular Fractionation and GDP-mannose Transport
Assay--
Exponentially growing yeast cells
(A600 = 2-3) were harvested, resuspended in
spheroplast buffer (1.0 M sorbitol, 50 mM Tris, pH 7.5, 2 mM magnesium chloride, 0.14% 2-mercaptoethanol)
and converted to spheroplasts by the addition of 5 units of
lyticase/A600 of cells, as described previously
(16). After washing twice with ice-cold spheroplast buffer,
spheroplasts from 500 A600 units of cells were
suspended in 10 ml of ice-cold lysis buffer (0.25 mM
sorbitol, 20 mM HEPES, pH 7.4, 50 mM potassium
acetate, 1 mM dithiothreitol) including a protease
inhibitor mixture (200 µM leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 0.1 mM
chloro-(4-tosylamido)7-amino-2-heptanone-HCl, 1 µM
pepstatin) and lysed by Dounce homogenization. After centrifugation at
3000 × g for 3 min to remove unbroken cells and
debris, the supernatant (S3) was collected and centrifuged at
17,000 × g for 15 min at 4 °C to yield a pellet
(P17) and a supernatant (S17). The S17 supernatant was centrifuged at
100,000 × g for 40 min to produce a Golgi-enriched
fraction (P100). The P100 fractions were resuspended into 200 µl of
buffer 88 (0.25 M sorbitol, 20 mM HEPES, pH
6.9, 150 mM potassium acetate, 5 mM magnesium
acetate), and their protein concentrations were determined by the BCA
reagent (Pierce).The GDP-mannose transport assay was performed
according to the procedure described by Roy et al. (28) with
the following modifications. 500 µg of the P100 fraction was
incubated in buffer 88 containing variable amounts of
GDP-[3H]mannose (180 cpm/pmol) at 30 °C for 5 min in a
final volume of 100 µl. Reactions were stopped by the addition of 1 ml of ice-cold buffer 88 and placed on ice. Diluted samples were
filtrated and washed with 15 ml of buffer 88 on 24-mm diameter,
0.45-µm pore size HA filters (Millipore Corp.). After air drying,
filters were dissolved in 1 ml of ethylene glycol methyl ether, and the
radioactivity was quantitated by scintillation counting. Counts bound
to membranes at time 0 were used to determine the amount of
GDP-[3H]mannose that nonspecifically bound to the membrane.
Mapping the Mutation in the vrg4-2 Allele Identifies a Motif That
Is Conserved among GDP-mannose Transporters--
Although a deletion
of the VRG4 gene is lethal, we previously isolated a viable
allele of vrg4 that has a severe glycosylation phenotype
both in vivo and in vitro. vrg4-2
strains are defective in glycoprotein and glycolipid glycosylation and
display a level of nucleotide sugar transport activity in
vitro that is about 25% reduced from those of wild type
strains (18). To identify the molecular basis for this mutant
phenotype, the sequence of the vrg4-2 mutant
allele was determined. The mutant gene was cloned by PCR (see
"Experimental Procedures"). A comparison of the DNA sequence from
the mutant and wild type VRG4 gene revealed a single C to A
base pair change at nucleotide position 857. This mutation results in
the replacement of an alanine with an aspartate at position 286, whose
location in the protein is shown graphically in the hydropathy plot in
Fig. 1A. This mutant protein
will be referred to as Vrg4-A286Dp. Three other viable missense
mutations in VRG4 with similar defects in glycosylation have
also been mapped to this region. The vig4-1
allele contains an alteration of the same Ala286, but to
Val; vig4-2 contains an amino acid substitution
of Ser278 to Cys (17). The van2-93
mutation replaces Arg274 with His (20). All of these amino
acids are clustered in a region of the protein spanning amino acids
271-291 that is highly conserved in other GDP-mannose transporters,
including those from Candida albicans, Candida
glabrata,2 and
Leishmania donovani (29), and putative NSTs from
Arabidopsis, rice, and Schizosaccharomyces pombe
that were identified through their strong sequence homology to Vrg4p
(Fig. 1B). The consensus motif surrounding
Ala286 is WXXXTTYSXVG(A/S)LNK(L/I)P.
We henceforth refer to this as the GALNK motif. An alignment of this
region from proteins closely related to Vrg4p and GDP-mannose
transporters (group I proteins) or to the UDP-sugar transporters (group
II proteins) is shown in Fig. 1B. The use of secondary
structure algorithms and hydropathy analysis predicts that Vrg4p
contains between 6 and 8 transmembrane-spanning domains. Since both the
N and C termini of Vrg4p face the cytoplasm (16), the GALNK-containing
region is predicted to span the junction between the penultimate
transmembrane-spanning domain and the preceding hydrophilic loop (see
Fig. 1A), with the conserved Ala286 just
abutting the lipid bilayer at the cytosolic face. Strikingly, when this
same predicted domain from the family of UDP-sugar transporters is
aligned to Vrg4p (based on hydropathy profiles), certain amino acids
are also invariantly conserved, including those aligning to
Thr281, Lys289, and Phe300 of
Vrg4p, but this GALNK motif is unique to Vrg4p and related GDP-mannose
transporters.
Mutations in the GALNK Motif Affect Normal Growth and
Viability--
To examine whether mutations in this conserved region
are important for Vrg4p function, site-directed mutagenesis was used to
alter the invariant amino acids in this region to Ala, except for
Gly285 and Lys289, which were changed to Ala or
Asp. The mutations that were introduced and their resulting phenotypes,
using each of the assays described below, are summarized in Table
I.
To determine whether these mutant alleles affect viability,
complementation of the lethality associated with loss of
VRG4 function was examined. Each of these mutant alleles was
introduced into a yeast strain containing VRG4 under the
control of the glucose-repressible GAL1 promoter. This
strain grows normally in the presence of galactose but fails to grow
when VRG4 gene expression is repressed in the presence of
glucose (16). Each mutant allele was expressed either on a low copy,
CEN-containing plasmid, under the control of the VRG4 promoter, or on a high copy, 2µ-containing plasmid,
under control of the TDH1 promoter. These strains were
plated on media containing either galactose or glucose as the sole
carbon source (Fig. 2). At low copy, most
of the mutant alleles could not support growth on glucose-containing
media, with the exceptions of the S282A and N288A alleles. Therefore,
at physiological levels, a mutation of most of these amino acids
results in a null phenotype (Fig. 2A). In contrast, when
expressed at high copy, only mutations at Gly285 and
Lys289 (and only when changed to Asp) were nulls, while
overexpression of the other mutant Vrg4 proteins could complement the
GAL1-VRG4-associated growth defect on glucose media (Fig.
2B). These results demonstrate that most mutations within
the GALNK motif cause a null phenotype when expressed at physiological
levels, but this effect can be overcome by high copy expression of
these alleles.
Mutations in the GALNK Motif of Vrg4 Do Not Affect Protein
Stability, Dimer Formation, or Golgi Localization--
To analyze
these mutant Vrg4 proteins, each mutant allele was tagged with either
the HA or myc epitope, appended to the C terminus (see "Experimental
Procedures"). To determine whether mutations in the GALNK motif of
Vrg4p alter protein stability, the steady state levels of these mutant
proteins were compared quantitatively with that of the wild type Vrg4
protein by Western blot analysis. Yeast strains were constructed that
coexpress wild type VRG4-HA3 and
VRG4-myc3 or each of the mutant vrg4-HA3 and vrg4-myc3 alleles.
Membrane proteins from each of these strains were solubilized with 1%
digitonin and analyzed by immunoblotting with anti-HA antibody. Shown
in Fig. 3A are representative
alleles that were found by complementation analyses to be either
partial or complete null alleles, i.e. G285D, A286D, and
K289A. By this assay, we found that the levels of the wild type and
mutant Vrg4-HA proteins were virtually identical. Similar results were
found for all of the mutations introduced into the GALNK motif (see
Table I), demonstrating that mutations in the GALNK motif of Vrg4p do
not affect protein stability and that the abnormal growth properties
observed in these mutants cannot be attributed to reduced Vrg4 protein
levels. Furthermore, these results imply that the high copy
complementation of growth by these vrg4 alleles results from
increased levels of mutant Vrg4p in vivo.
Vrg4p normally functions as a dimer. Dimer formation, which is mediated
by the C-terminus of the protein, is essential for its
in vivo function (16, 17). To determine whether proteins with mutations in the GALNK motif were affected in their oligomer assembly properties, a coimmunoprecipitation assay was used. The same
detergent extracts used in the Western blot assay described above were
incubated with the anti-HA monoclonal antibody, using conditions that
we previously demonstrated maintain active dimers (16). To measure the
relative amount of myc-tagged protein that associated with the
HA-tagged Vrg4 protein, proteins precipitated with anti-HA antibody
were fractionated by SDS-PAGE and immunoblotted with anti-myc rabbit
antiserum (Fig. 3B). Comparable amounts of myc-tagged Vrg4p,
Vrg4-G285D-myc, Vrg4-A286D-myc, and Vrg4-K289A-myc precipitated with
the corresponding HA-tagged Vrg4p (Fig. 3B, compare
lane 2 with lanes 3,
4, and 5). These results indicated that amino
acids 285, 286, and 289 are essential for normal growth and viability,
but not for oligomer assembly.
The Vrg4 protein is localized to the yeast Golgi, and its localization
to this compartment is essential for its function (16, 18). To test
whether mutations within the GALNK motif may affect transport to the
Golgi, the intracellular localization of each mutant protein was
compared with the normal Vrg4 protein. Cells expressing the
VRG4-HA or vrg4-G285D-HA, vrg4-A286D-HA, and
vrg4-K289A-HA alleles were fixed with formaldehyde, and the
HA-tagged proteins were detected by indirect immunofluorescence using
antibodies directed against the HA-epitope. By this assay, each of
these mutant proteins displayed the same punctate pattern
characteristic of the yeast Golgi that is observed for the wild type
Vrg4 protein and Och1p, a resident Golgi mannosyltransferase (Fig.
3C), suggesting that the mutant proteins are correctly
localized to the Golgi. Therefore, the inactivity of Vrg4 proteins with
mutations in the GALNK motif is not due to their mislocalization.
Vrg4p with a Mutation in the GALNK Motif Has Reduced Transport
Activity in Vitro That Can Be Suppressed by Increasing Substrate
Concentration--
The specificity of this GALNK motif to several NSTs
that are known to transport GDP-mannose, together with the data that
show these mutations do not affect protein stability, dimerization, or
localization, prompted us to test the idea that this region of Vrg4 may
play a role in binding to the nucleotide sugar or nucleoside
monophosphate. Binding to the nucleotide sugar was the preferred model,
since this domain is predicted to reside in the cytosol (16). We
previously demonstrated that the vrg4-2 (i.e. the vrg4-A286D) allele displays a defect in
luminal GDP-mannose uptake, using an in vitro permeabilized
cell system. Under standard conditions, including GDP-mannose at a
final concentration of 3 µM, the vrg4-A286D
strain is about 25-fold reduced in luminal GDP-mannose uptake activity,
compared with the isogenic VRG4 strain (18). To examine the
kinetic parameters and substrate dependence of this transport defect in
a more purified system, Golgi-enriched membranes were prepared from
isogenic strains containing either the VRG4 or
vrg4-A286D alleles. A similar difference in the transport efficiency of the vrg4-A286D and VRG4 strains was
observed in this system under standard conditions as in permeabilized
cells (data not shown). The apparent Km for this
reaction was found to be about 3 and 20 µM for the
VRG4 and vrg4-A286D strain, respectively.
However, transport into these vesicles was saturable at similar
concentrations of GDP-mannose, with Vmax values
of about 120 and 140 pmol/mg/5 min for the mutant and wild type strain, respectively (Fig. 4). In other words,
increasing the substrate concentration in vitro enabled a
suppression of the transport defect of the vrg4-A286D
strain.
Photolabeling of Vrg4-HAp to Guanosine
5'-[ Vrg4p with a Mutation in the GALNK Motif Is Defective in Binding to
Guanosine 5'-[ Through our analyses of the vrg4-2 mutant
allele, we identified a phylogenetically conserved motif in Vrg4p that
is required for nucleotide sugar transport activity. Having ruled out a
requirement of the amino acids in this region for protein stability,
localization to the Golgi, or protein multimerization, we propose a
model in which this region of the protein is required for binding to
GDP-mannose. This model is based on the specificity of this motif to
GDP-mannose transporters and by the induced phenotype suffered by
vrg4 mutants containing alterations within this motif. The
observations that increasing either Vrg4 protein (in vivo)
or substrate concentration (in vitro) overcome these
vrg4 mutant phenotypes and that at least two amino acids in
this motif, Ala286 and Gly285, are required for
maximal binding to the photoprobe, [ The "GALNK" consensus motif surrounding Ala286 is
TTYSXVG(A/S)LNK(L/I)P. While most of the mutations that were
introduced into this region resulted in a null phenotype at low copy,
when overexpressed, these mutant alleles could complement the lethality
caused by loss of VRG4 function. High copy complementation
of conditional loss of VRG4 function appears to be specific
to this class of vrg4 alleles. For instance, mutations that
delete the C terminus, which regulates Vrg4p dimer formation, or the N
terminus, which is required for ER export to the Golgi, also result in
a null phenotype. In contrast to the GALNK mutants, overexpression of vrg4 The specificity of this GALNK motif to several NSTs that are known to
transport GDP-mannose (e.g. the S. cerevisiae and
Candida Vrg4 proteins and the Leishmania Lpg2p)
and its absence from UDP-sugar transporters are also consistent with a
role for this motif in binding to this nucleotide sugar. The GALNK
consensus motif surrounding Ala286 was also found in
several plant ORFs identified through their strong sequence similarity
to Vrg4p. While there is little evidence for the terminal addition of
mannose on plant glycoproteins, luminal GDP-mannose may be a required
substrate for the intracellular synthesis of the hemicelluloses,
glucomannan and galactomannan. The cellular compartment in which
these polysaccharides are synthesized has not been determined. However,
the galactosyltransferase that catalyzes the addition of galactose onto
the mannose backbone has all of the hallmarks of a Golgi-localized,
type II membrane protein (31), suggesting that galactomannan, like
xyloglucan, may be synthesized within the lumen of the plant Golgi.
Two S. cerevisiae proteins, Yel004p and Ypl244c, were
identified as members of the UDP-sugar transporter family that do not contain the conserved GALNK motif. Yel004p (also known as Yea4p) has
recently been characterized as an ER-localized UDP-GlcNAc transporter
(32). The function of Ypl244c is unknown, although this protein is most
similar to the S. pombe UDP-Gal transporter and appears to
be localized in the Golgi (16). UDP-galactose transport in membrane
fractions from S. cerevisiae has been reported (28),
although its physiological relevance is unclear. It may be that
galactose is a rare sugar modification in S. cerevisiae or
that this activity is derived from a transporter with a broader substrate specificity, for instance a UDP-glucose transporter. There is
evidence that glucan-containing polymers required for cell wall
-32P]triphosphate
-azidoanilide, a photoaffinity substrate analogue whose binding to
Vrg4-HAp was specifically inhibited by GDP-mannose. Taken together,
these data are consistent with the model that amino acids in this
region of the yeast GDP-mannose transporter mediate the recognition of
or binding to nucleotide sugar prior to its transport into the Golgi.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]GTP-azidoanilide, implicating this domain
in the mediation of substrate binding.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ura3-52 leu2-211
vrg4-2) (10) was used as the source of genomic DNA for
the cloning of the vrg4-2 allele. Its isogenic
parental strain is RSY255 (MAT
ura3-52
leu2-211). XGY13 is a derivative of RSY255 but contains an
HA-tagged allele of VRG4 integrated at the
ura3-52 locus, driven by the TPI promoter (16).
XGY14 was constructed from W303a (MATa ade2-1 ura3-1 his3-11
trp1-1 leu2-3, 112 can1-100) in which VRG4 is under
the control of the GAL1 promoter. XGY15 and XGY16 are
derivatives of XGY14 but contain, in addition, a TPI-driven
HA-tagged allele of VRG4 or vrg4-A286D, respectively, integrated at the ura3-52 locus. JPY26-3d
(MAT
ura3-52 leu2-211 his3 dpm1
vrg4-2) or the VRG4 isogenic parental strain, JPY25-6c (MAT
ura3-52 leu2-211 his3
dpm1) (18) were used for GDP-mannose transport assays.
RHL-HA3 (18) that contains the
corresponding 3'-end of the VRG4 gene with the triple HA
tag. This plasmid, pRSvrg4-A286D-HA3, encodes the
vrg4-A286D mutant protein tagged with three copies of the HA
epitope at the C terminus in pRS316. Using the same strategy,
pRSvrg4-A286D-myc3 was constructed by replacement of the
MfeI/SacI fragment of pRSvrg4-A286D with that
from SK
VRG4-myc3 (16), which has three copies
of the myc epitope at the C terminus. To construct
pRS315vrg4-A286D-myc3, a HindIII/SacI fragment from pRSvrg4-A286D-myc3 containing the entire
vrg4-A286D-myc3 ORF and VRG4 promoter
sequences was subcloned into pRS315, a CEN6/LEU2 vector
(24). Similarly, pRS315-VRG4-myc3 was generated by
subcloning a HindIII/SacI fragment from
pRS316VRG4-myc3 (16) into pRS315.
(Stratagene) to generate
pSK
VRG4-HA3. This
SK
VRG4-HA3 plasmid was used as a template for
site-directed mutagenesis, using the QuikChangeTM
site-directed mutagenesis kit (Stratagene). Mutagenic primers were
designed to change Thr280, Ser282,
Gly285, Leu287, Asn288,
Lys289, or Pro291 to alanine and the
Gly285 or Lys289 to aspartate. This generated a
series of mutant vrg4 alleles in pBluescript
SK
, including
pSK
vrg4-T280A-HA3,
pSK
vrg4-S282A-HA3,
pSK
vrg4-G285A-HA3,
pSK
vrg4-G285D-HA3,
pSK
vrg4-L287A-HA3,
pSK
vrg4-N288A-HA3,
pSK
vrg4-K289A-HA3,
pSK
vrg4-K289D-HA3, and
pSK
vrg4-P291A-HA3. All of these mutations
were confirmed by DNA sequence analysis.
mutant vrg4 plasmid series. This generated
a plasmid series in which each of the mutant HA-tagged vrg4
alleles was cloned in pRS316, including pRSvrg4-T280A-HA3,
pRSvrg4-S282A-HA3, pRSvrg4-G285A-HA3, pRSvrg4-G285D-HA3, pRSvrg4-L287A-HA3,
pRSvrg4-N288A-HA3, pRSvrg4-K289A-HA3, pRSvrg4-K289D-HA3, and pRSvrg4-P291A-HA3. Some
of these mutant vrg4 alleles were also tagged with the myc
epitope. These plasmids were constructed by replacing the 3'-end of
each vrg4 mutant allele with the corresponding
MfeI/XbaI fragment from
pSK-VRG4-myc3. The resulting plasmids
contain the myc-tagged mutant vrg4 alleles in pRS316. To
place these genes in a CEN6/LEU2 vector, a 1.3-kb
HindIII/XbaI fragment containing the entire
mutant vrg4-myc3 ORF and VRG4 promoter
sequences was subcloned into pRS315 (24) to generate
pRS315vrg4-G285D-myc3 and
pRS315vrg4-K289A-myc3.
VRG4-HA or pSK
vrg4-HA,
containing just the ORF was cloned into pT
O (26), which places their
expression under the constitutive triose-phosphate isomerase
(TPI) promoter. Linearization of these plasmids with XhoI within the URA3 gene targets integration at
the ura3-52 locus
-32P]GTP-azidoanilide Binding
Assay--
Permeabilized yeast cells were prepared as described (18).
Permeabilized yeast cells were quickly thawed at 37 °C and washed three times with 1.0 ml of ice-cold buffer A (20 mM Hepes
(pH 6.8), 150 mM KoAc, 250 mM sorbitol) by a
quick pulse in a microcentrifuge. Pelleted cells were resuspended in
buffer A to 1/2 their original volume. Each reaction contained
40 µl of concentrated permeabilized yeast cells (about 7 A600 units), to which was added guanosine 5'-[
-32P]triphosphate
-azidoanilide (Affinity
Labeling Inc., Lexington, KY) to a final concentration of 5 µM. Each reaction was incubated with the photo probe at
room temperature for 1 min before exposing to short wave UV light,
using a hand-held UV lamp (Photodyne, CA) for 90 s. The reaction
was quenched by the addition of 2 µl of 0.5 M
dithiothreitol, and samples were placed on ice. Free radioactive
solutes were removed by washing the cells three times with 1 ml of
ice-cold buffer A. Proteins were solubilized with 500 µl of ice cold
1% Triton X-100 in PBS and centrifuged for 3 min at 14,000 × g to remove debris. For the visualization of 32P-labeled proteins, 20 µl of this Triton X-100 extract
was directly applied to SDS-PAGE. Alternatively, aliquots were
immunoprecipitated by the addition of anti-HA antibody and protein
A-Sepharose for 1-2 h. The precipitated protein was washed with PBS
plus 1% Triton three times, resuspended in SDS-PAGE sample buffer,
heated at 55 °C, and fractionated by 10% SDS-PAGE. For visualizing
32P-labeled proteins, polyacrylamide gels were dried down
and directly exposed to x-ray film. For immunodetection of Vrg4-HA,
proteins were transferred from the polyacrylamide gel to polyvinylidene difluoride membranes (Millipore Corp.) and detected by Western immunoblotting using anti-HA antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The vrg4-2
allele contains a single mutation (A286D) in a region of the
protein that is highly conserved among other NSTs. A, a
hydropathy profile of the Vrg4 protein, with the location of the A286D
mutation indicated with a circle. B, an alignment
of the region surrounding Ala286 (depicted with an
asterisk) in other nucleotide sugar transporters. Group I
proteins are closely related to the GDP-mannose transporters defined by
the L. donovani Lpg2 protein and the S. cerevisiae and C. albicans Vrg4 proteins. Group II
proteins are most highly related to UDP-sugar transporters. The
accession numbers of related but uncharacterized ORFs are indicated.
The identification of these proteins was obtained using BLAST version
2.0 (38). Alignments were performed using the DNASTAR MegAlign program,
using the Clustal algorithm. The consensus was stringently defined as a
majority of six out of seven identical residues for group I proteins
(shaded in black) and five out of eight identical
residues for group II (shaded in gray). Residues
in group II proteins that are identical to those in group I are
shaded in black.
Phenotypes of vrg4 "GALNK" mutants
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Fig. 2.
Amino acids in the Vrg4p GALNK motif are
essential for normal growth. Strain XGY14, containing
VRG4 under control of the glucose-repressible
GAL1 promoter was transformed with either
CEN-containing plasmids (A) or 2µ-containing
plasmids (B) harboring the VRG4 or mutant
vrg4 alleles containing single missense mutations that
affect amino acids within the GALNK motif (as indicated) and streaked
onto YPA medium supplemented with galactose or glucose.
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Fig. 3.
Mutations in the GALNK motif of Vrg4p do not
affect protein stability, dimer formation, or Golgi localization.
A, Western blot of the Vrg4-HAp and those containing
mutations within the GALNK motif. Digitonin lysates were prepared from
wild type yeast (SEY6210) expressing VRG4-myc alone
(lane 1), coexpressing both
VRG4-HA3 and VRG4-myc3
(lane 2), both vrg4-G285D-HA3
and vrg4-G285D-myc3 (lane 3),
both vrg4-A286D-HA3 and
vrg4-A286D-myc3 (lane 4), or
both vrg4-K289A-HA3 and
vrg4-K289A-myc3 (lane 5).
Plasmid-borne HA-tagged alleles were expressed in a pRS316 vector,
while myc-tagged alleles were in a pRS315 vector (see Table
II). After loading equivalent amounts of
protein per well, proteins were fractionated by SDS-PAGE and subjected
to Western blot analysis with anti-HA antibodies and detected by
chemiluminescence. B, the same extracts used in the
experiment shown in A were immunoprecipitated
(IP) with anti-HA antibodies to isolate Vrg4-HAp-containing
protein complexes. After loading equivalent amounts of protein in each
lane and fractionating by SDS-PAGE, precipitates were immunoblotted
with anti-myc antibodies and detected by chemiluminescence.
C, indirect immunofluorescence of SEY6210 expressing
VRG4-HA (pRS316VRG4-HA3) or the mutant alleles
vrg4-G285D-HA, vrg4-A286D-HA, and
vrg4-K289A-HA (in pRS316-based vectors). Also shown for
comparison is the localization of the Golgi marker, Och1-HAp. Fixed
cells were treated with anti-HA antibodies, followed by fluorescein
isothiocyanate-conjugated anti-mouse IgG.
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Fig. 4.
Substrate concentration-dependence of
GDP-mannose transport in membrane vesicles from VRG4
and vrg4-A286D strains. A P100 fraction
(0.5 mg of protein) prepared from the vrg4-A286D strain,
JPY26-3d, was mixed with varying concentrations of
GDP-[3H]mannose (180 cpm/pmol) in a final volume of 100 µl. After incubation for 5 min at 30 °C, the transport activity
was measured as described under "Experimental Procedures." The
average values of transport activity (pmol of
GDP-[3H]mannose/mg of protein/5-min reaction) plus S.D.,
shown by error bars, from three individual
experiments are plotted. Inset,
GDP-[3H]mannose transport activity was measured in
parallel in a P100 fraction prepared from the isogenic VRG4
strain, JPY25-6d.
-32P]Triphosphate
-Azidoanilide--
We sought
to further test the model that mutations in the GALNK domain affect
substrate binding by developing an assay for GDP-mannose binding. Since
an effective competitive inhibitor of GDP-fucose transport is
8-azidoguanosine-5'-triphosphate (30), this and other commercially
available azido-[32P]GTP or [32P]GDP
photoaffinity probes were examined for their ability to bind to Vrg4p
with specificity. By assaying a number of different photoaffinity
probes, we found that guanosine 5'-[
-32P]triphosphate
-azidoanilide ([
-32P]GTP
AA) bound to Vrg4-HAp
with high specificity in a light-dependent manner, but in
contrast to what has been reported for the GDP-fucose transporter,
8-azido-GTP did not (data not shown). To mimic conditions in which
GDP-mannose normally binds to Vrg4p, permeabilized cells were prepared
from a yeast strain that expresses an HA-tagged allele of
VRG4, using the same protocol that was previously shown to
promote in vitro luminal transport of GDP-mannose (18).
After extensive washing to deplete cytosolic GDP-mannose, these
membranes were incubated with [
-32P]GTP
AA, in the
presence or absence of UV light. To reduce potential binding to other
GTP-binding proteins, magnesium, which is not a required cofactor for
GDP-mannose transport, was omitted from the incubation buffer. After
photolabeling, membrane proteins were solubilized with 1% Triton, and
aliquots were either directly fractionated by SDS-PAGE to examine the
complete constellation of 32P labeled proteins or subjected
to immunoprecipitation with anti-HA antibodies to examine Vrg4-HAp.
Under these conditions, Vrg4-Hap could be radiolabeled specifically in
a light-dependent manner, as demonstrated by the
comigration of affinity-purified 32P-Vrg4-HAp with a
protein of the same molecular weight in protein extracts that is
notably absent in cells that do not express the HA-tagged allele (Fig.
5A). To test the specificity
of cross-linking, photolabeling reactions were preincubated with
GDP-mannose at concentrations ranging from 2- to 20-fold molar excess
over [
-32P]GTP
AA. Radiolabeling of Vrg4-HAp with
[
-32P]GTP
AA was inhibited by the presence of 10 µM GDP-mannose, but no reduction was seen when
UDP-glucose was present in the reaction, even at 100 µM
(Fig. 5B), suggesting that GDP-mannose is a competitive inhibitor of [
-32P]GTP
AA.
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Fig. 5.
Photolabeling of Vrg4p with guanosine
5'-[ -32P] triphosphate
-azidoanilide. Permeabilized yeast cells were
prepared from a wild type yeast strain (RSY255) or its isogenic
derivative that contains an additional, integrated HA-tagged allele of
VRG4 (XGY13). 5 A600 units of
permeabilized cells were incubated with the photoaffinity probe,
[
-32P]GTP
AA, cross-linked by exposure to UV light,
and solubilized with 1% Triton X-100, as described under
"Experimental Procedures." A, aliquots of these
detergent extracts were fractionated directly by SDS-PAGE
(lane 1, XGY13; lane 2,
RSY255) or immunoprecipitated (IP) with anti-HA antibody.
The HA-immunoprecipitated proteins were suspended in sample buffer and
applied to SDS-PAGE (lane 3, RSY255;
lane 4, XGY13). The polyacrylamide gel was
dried down and directly exposed to x-ray film for 24 h.
B, permeabilized cells were incubated with the photoaffinity
probe, [
-32P]GTP
AA, after a preincubation with
varying amounts of GDP-mannose and UDP-glucose (as indicated),
cross-linked by exposure to UV light, and solubilized with 1% Triton
X-100. Proteins were immunoprecipitated with anti-HA antibody were
fractionated by SDS-PAGE, and [32P]Vrg4-HAp was detected
by exposing the dried gel to x-ray film for 24 h.
-32P]Triphosphate
-Azidoanilide--
To determine whether a mutation in the GALNK
motif affects the efficiency of radiolabeling with
[
-32P]GTP
AA, the level of Vrg4-A286D-HAp that could
be cross-linked was compared with the wild type protein. Since Vrg4p
forms dimers in vivo and in vitro, our strategy
was to establish conditions in which photolabeling of mutant Vrg4p
homodimers could be compared with wild type Vrg4p homodimers. Yeast
strains were constructed that contain the glucose-repressible
GAL1-VRG4 gene and an additional copy of either
VRG4-HA or vrg4-A286D-HA, integrated at the
ura3-52 locus and driven by the strong, constitutive
TPI promoter. After growth in galactose, these strains were
shifted into medium supplemented with glucose to turn off the
expression of the endogenous VRG4 gene and in so doing
minimize the ratio of heterodimers formed between Vrg4p and the
HA-tagged mutant or wild type Vrg4 proteins. As a control,
permeabilized cells were also prepared from the parental strain that
contains only the GAL1-VRG4 gene that was grown in medium
supplemented with galactose. After incubation with
[
-32P]GTP
AA, membrane proteins from each of these
strains were solubilized with 1% Triton X-100, immunoprecipitated with
anti-HA antibody, and fractionated by SDS-PAGE. Western blot analysis
indicated that equivalent amounts of the mutant and wild type
Vrg4-HA-tagged proteins were present in each sample (Fig.
6A). In contrast, analysis of
the levels of 32P-labeled HA-tagged proteins in these
samples, shown graphically in Fig. 6B, indicated a
significant decrease in the presence of photolabeled Vrg4-A286D-HAp
compared with the wild type Vrg4-HAp. Quantitation of these results by
densitometry, averaged over three experiments, demonstrated this
reduction to be ~30% of wild type. Analysis of the
vrg4-G285D-HA3 allele in a wild type strain
background demonstrated that a mutation at Gly285 also
reduced cross-linking of Vrg4-G285D-HA3 to
[
-32P]GTP
AA (data not shown). The relatively modest
decrease in photolabeling, compared with the 25-fold decreased
transport defect in this mutant may be due to the presence of residual
Vrg4p that forms heterodimers consisting of mutant and wild type Vrg4p
monomers, although this possibility was not further tested. However,
these data demonstrate that a mutation of Ala286 and
Gly285 cause a reduction in the binding of Vrg4p to this
photoaffinity probe.
Plasmids used in this study
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Fig. 6.
Vrg4-A286D-HAp is defective in binding
guanosine
5'-[ -32P]triphosphate
-azidoanilide. Yeast strains containing the
GAL1-VRG4 gene (XGY14) and its derivatives, which contain an
additional integrated copy of VRG4-HA3
(XGY15) or vrg4-A286D-HA3 (XGY16), were
shifted to growth in YPA+ glucose medium and used to prepare
permeabilized cells for photolabeling. Permeabilized cells were also
prepared from XGY14 grown in a YPA medium with galactose
(lanes 3 and 6). Permeabilized cells
were incubated with [
-32P]GTP
AA, cross-linked by
exposure to UV light, solubilized with 1% Triton X-100, and
immunoprecipitated with anti-HA antibodies, and the precipitated
proteins were resuspended in SDS-PAGE sample buffer as described under
"Experimental Procedures." A, aliquots of
immunoprecipitates were subjected to Western blot analysis using
anti-HA antibodies and detected by chemiluminescence to compare the
amount of HA-tagged protein in each sample (lanes
1-3). B, aliquots of each of sample were also
fractionated by SDS-PAGE, and the gels were dried and directly exposed
to x-ray film to compare the relative amounts of
32P-labeled HA-tagged proteins (lanes
4-6). C, the relative amount of
32P-labeled Vrg4-HA proteins in each sample was quantitated
by densitometry, and values are shown graphically, with the binding
activity of wild type Vrg4-HA protein complex set as 100%.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]GTP-azido
anilide, are consistent with a direct role of the GALNK motif in
binding GDP-mannose.
N null alleles leads to a dominant
negative phenotype, while overexpression of
vrg4
C alleles has no affect (16). Since none of the GALNK mutations affect Vrg4 protein stability, we infer that
overexpression of these vrg4 alleles results in increased intracellular levels of the encoded proteins. One interpretation of
this high copy complementation is that the amino acids that are altered
in these mutant Vrg4 proteins are important for substrate interaction.
Alteration of Thr280, Ala286,
Leu287, and Asn288 may cause a reduced affinity
for GDP-mannose, but this effect can be overcome by increased mutant
Vrg4p levels when these alleles are overexpressed. This interpretation
is greatly strengthened by our observation that the transport defect of
the A286D mutation can also be suppressed in vitro by
increasing the substrate concentration. vrg4 alleles
containing mutations in Lys289, Gly285, or
Pro291 resulted in loss of function at both low and high
copy levels of expression, which may indicate that these amino acids
are essential for binding GDP-mannose. An alternative explanation that
we have not ruled out is that residues in the GALNK motif do not
directly interact with nucleotide sugar but rather function to promote a conformation in the protein that is required for optimal substrate recognition and binding. Since Pro and Gly residues are particularly adept as "helix breakers," the phenotypes of the Gly285
or Pro291 mutants are consistent with this latter notion
that the structural integrity of the penultimate transmembrane-spanning
domain, abutting these residues, is required for binding to nucleotide
sugar. Since Vrg4p is essential and functions only as a dimer, a direct
assessment of the binding affinity of these mutant homodimers is
difficult, since these mutant vrg4 strains containing an
HA-tagged Vrg4p protein expressed at physiological levels are inviable.
However, in either case, the reproducible, although relatively modest, defect in binding to the photoaffinity probe by Vrg4-A286D-HAp (Fig. 6)
and Vrg4-G285D-HAp (not shown) argues that residues in the GALNK region
are required for binding GDP-mannose.
-1,6-glucan begin within the Golgi and/or the ER (33-36), implying
a requirement for luminal UDP-glucose. Transport of UDP-glucose into
the ER has been demonstrated in S. cerevisiae (37), but no
candidate gene product that mediates this transport event has yet been
identified. It is tempting to speculate that, as has been demonstrated
for Yel004p, Ypl244p may also function in the transport of a UDP-sugar
required for the intracellular synthesis of a cell wall carbohydrate
polymer, a possibility that we are currently testing.
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ACKNOWLEDGEMENTS |
---|
We thank Janet Leatherwood, Nancy Hollingsworth, Tadashi Suzuki, and Hiroyuki Tachikawa for valuable advice and discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM48467 (to N. D.)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. Tel.: 631-632-9309;
Fax: 631-632-8575; E-mail: Neta.Dean@sunysb.edu.
Published, JBC Papers in Press, November 6, 2000, DOI 10.1074/jbc.M009114200
2 A. Nishikawa, J. Poster, and N. Dean, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NST, nucleotide
sugar transporter;
HA, hemagglutinin;
kb, kilobase pair(s);
PCR, polymerase chain reaction;
ORF, open reading frame;
PAGE, polyacrylamide gel electrophoresis;
[-32P]GTP
AA, guanosine 5'-[
-32P]triphosphate
-azidoanilide;
ER, endoplasmic reticulum.
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