Identification of a Conserved Motif in the Yeast Golgi GDP-mannose Transporter Required for Binding to Nucleotide Sugar*

Xiao-Dong Gao, Akiko Nishikawa, and Neta DeanDagger

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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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'-[gamma -32P]triphosphate gamma -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, [gamma -32P]GTP-azidoanilide, implicating this domain in the mediation of substrate binding.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (MATalpha 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 (MATalpha 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 (MATalpha ura3-52 leu2-211 his3 dpm1 vrg4-2) or the VRG4 isogenic parental strain, JPY25-6c (MATalpha ura3-52 leu2-211 his3 dpm1) (18) were used for GDP-mannose transport assays.

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-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.

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- (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.

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- 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.

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-VRG4-HA or pSK-vrg4-HA, containing just the ORF was cloned into pTalpha 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

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).

[gamma -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'-[gamma -32P]triphosphate gamma -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.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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.



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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.

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.


                              
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Table I
Phenotypes of vrg4 "GALNK" mutants

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.



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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.

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.



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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.

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.



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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.

Photolabeling of Vrg4-HAp to Guanosine 5'-[gamma -32P]Triphosphate gamma -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'-[gamma -32P]triphosphate gamma -azidoanilide ([gamma -32P]GTPgamma 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 [gamma -32P]GTPgamma 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 [gamma -32P]GTPgamma AA. Radiolabeling of Vrg4-HAp with [gamma -32P]GTPgamma 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 [gamma -32P]GTPgamma AA.



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Fig. 5.   Photolabeling of Vrg4p with guanosine 5'-[gamma -32P] triphosphate gamma -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, [gamma -32P]GTPgamma 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, [gamma -32P]GTPgamma 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.

Vrg4p with a Mutation in the GALNK Motif Is Defective in Binding to Guanosine 5'-[gamma -32P]Triphosphate gamma -Azidoanilide-- To determine whether a mutation in the GALNK motif affects the efficiency of radiolabeling with [gamma -32P]GTPgamma 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 [gamma -32P]GTPgamma 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 [gamma -32P]GTPgamma 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.


                              
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Table II
Plasmids used in this study



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Fig. 6.   Vrg4-A286D-HAp is defective in binding guanosine 5'-[gamma -32P]triphosphate gamma -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 [gamma -32P]GTPgamma 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

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, [gamma -32P]GTP-azido anilide, are consistent with a direct role of the GALNK motif in binding GDP-mannose.

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 vrg4Delta N null alleles leads to a dominant negative phenotype, while overexpression of vrg4Delta 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.

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 beta -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.


    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.

Dagger 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; [gamma -32P]GTPgamma AA, guanosine 5'-[gamma -32P]triphosphate gamma -azidoanilide; ER, endoplasmic reticulum.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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