Distinct Protein Domains of the Yeast Golgi GDP-mannose Transporter Mediate Oligomer Assembly and Export from the Endoplasmic Reticulum
2000; Elsevier BV; Volume: 275; Issue: 23 Linguagem: Inglês
10.1074/jbc.m909946199
ISSN1083-351X
Autores Tópico(s)Fungal and yeast genetics research
ResumoThe substrates for glycan synthesis in the lumen of the Golgi are nucleotide sugars that must be transported from the cytosol by specific membrane-bound transporters. The principal nucleotide sugar used for glycosylation in the Golgi of the yeastSaccharomyces cerevisiae is GDP-mannose, whose lumenal transport is mediated by the VRG4 gene product. As the sole provider of lumenal mannose, the Vrg4 protein functions as a key regulator of glycosylation in the yeast Golgi. We have undertaken a functional analysis of Vrg4p as a model for understanding nucleotide sugar transport in the Golgi. Here, we analyzed epitope-tagged alleles of VRG4. Gel filtration chromatography and co-immunoprecipitation experiments demonstrate that the Vrg4 protein forms homodimers with specificity and high affinity. Deletion analyses identified two regions essential for Vrg4p function. Mutant Vrg4 proteins lacking the predicted C-terminal membrane-spanning domain fail to assemble into oligomers (Abe, M., Hashimoto, H., and Yoda, K. (1999)FEBS Lett. 458, 309–312) and are unstable, while proteins lacking the N-terminal cytosolic tail are stable and multimerize efficiently, but are mislocalized to the endoplasmic reticulum (ER). Fusion of the N terminus of Vrg4p to related ER membrane proteins promote their transport to the Golgi, suggesting that sequences in the N terminus supply information for ER export. The dominant negative phenotype resulting from overexpression of truncated Vrg4-ΔN proteins provides strong genetic evidence for homodimer formation in vivo. These studies are consistent with a model in which Vrg4p oligomerizes in the ER and is subsequently transported to the Golgi via a mechanism that involves positive sorting rather than passive default. The substrates for glycan synthesis in the lumen of the Golgi are nucleotide sugars that must be transported from the cytosol by specific membrane-bound transporters. The principal nucleotide sugar used for glycosylation in the Golgi of the yeastSaccharomyces cerevisiae is GDP-mannose, whose lumenal transport is mediated by the VRG4 gene product. As the sole provider of lumenal mannose, the Vrg4 protein functions as a key regulator of glycosylation in the yeast Golgi. We have undertaken a functional analysis of Vrg4p as a model for understanding nucleotide sugar transport in the Golgi. Here, we analyzed epitope-tagged alleles of VRG4. Gel filtration chromatography and co-immunoprecipitation experiments demonstrate that the Vrg4 protein forms homodimers with specificity and high affinity. Deletion analyses identified two regions essential for Vrg4p function. Mutant Vrg4 proteins lacking the predicted C-terminal membrane-spanning domain fail to assemble into oligomers (Abe, M., Hashimoto, H., and Yoda, K. (1999)FEBS Lett. 458, 309–312) and are unstable, while proteins lacking the N-terminal cytosolic tail are stable and multimerize efficiently, but are mislocalized to the endoplasmic reticulum (ER). Fusion of the N terminus of Vrg4p to related ER membrane proteins promote their transport to the Golgi, suggesting that sequences in the N terminus supply information for ER export. The dominant negative phenotype resulting from overexpression of truncated Vrg4-ΔN proteins provides strong genetic evidence for homodimer formation in vivo. These studies are consistent with a model in which Vrg4p oligomerizes in the ER and is subsequently transported to the Golgi via a mechanism that involves positive sorting rather than passive default. nucleotide sugar transporter(s) tobacco etch virus polymerase chain reaction open reading frame hemagglutinin phenylmethylsulfonyl fluoride fast protein liquid chromatography polyacrylamide gel electrophoresis endoplasmic reticulum untranslated region transmembrane domain(s) The Golgi complex serves as the intracellular site for the terminal carbohydrate modifications of proteins and lipids. These modifications are essential for life and play a variety of important biological roles, from protein folding to the regulation of cell surface properties. The substrates for the carbohydrate modification of both glycoproteins and glycolipids in the Golgi are nucleotide sugars, whose site of synthesis is the cytosol. These molecules must be transported into the Golgi lumen by membrane-bound nucleotide sugar transporters (NSTs)1 to be utilized by the glycosyltransferases. The current model for the transport of nucleotide sugars by the NSTs involves a one-for-one exchange reaction, in which the lumenal transport of a nucleotide sugar from the cytoplasm is coupled to the equimolar exit of the corresponding nucleotide monophosphate (2.Hirschberg C.B. Snider M.D. Annu. Rev. Biochem. 1987; 56: 63-87Crossref PubMed Scopus (443) Google Scholar, 3.Capasso J.M. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7051-7055Crossref PubMed Scopus (110) Google Scholar, 4.Waldman B.C. Rudnick G. Biochemistry. 1990; 29: 44-52Crossref PubMed Scopus (59) Google Scholar). The nucleotide monophosphate is generated through the action of the glycosyltransferases and nucleoside diphosphatases in the lumen of the Golgi. As a consequence of their role in substrate provision, the NSTs play an indispensable role in glycoconjugate synthesis, best evidenced by the severe phenotype of mutants with defects in Golgi transport of nucleotide sugars (for review, see Refs. 5.Hirschberg C.B. Robbins P.W. Abeijon C. Annu. Rev. Biochem. 1998; 67: 49-69Crossref PubMed Scopus (309) Google Scholar and 6.Kawakita M. Ishida N. Miura N. Sun-Wada G.H. Yoshioka S. J. Biochem. (Tokyo). 1998; 123: 777-785Crossref PubMed Scopus (54) Google Scholar). Many NST activities have been reported, which differ from one another in their substrate specificity. The diversity of glycosylation reactions in the mammalian Golgi requires the transport of many different nucleotide sugars and a correspondingly large number of NSTs. In contrast, the vast majority of carbohydrate modifications in the yeast Golgi are restricted to mannose additions that utilize GDP-mannose as the nucleotide sugar substrate (7.Abeijon C. Orlean P. Robbins P.W. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6935-6939Crossref PubMed Scopus (133) Google Scholar). In the yeast,Saccharomyces cerevisiae, lumenal GDP-mannose transport requires the VRG4 gene product (8.Dean N. Zhang Y.B. Poster J.B. J. Biol. Chem. 1997; 272: 31908-31914Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Mutations in this gene lead to a loss of nucleotide sugar transport in vitro and the underglycosylation of glycoproteins and glycosphingolipids in vivo. A deletion of the VRG4 gene is lethal, demonstrating that mannosylation in the Golgi is essential (9.Poster J.B. Dean N. J. Biol. Chem. 1996; 271: 3837-3845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Its strong homology to the Leishmania GDP-mannose transporter as well as to a large number of other NSTs (8.Dean N. Zhang Y.B. Poster J.B. J. Biol. Chem. 1997; 272: 31908-31914Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 10.Ma D. Russell D.G. Beverley S.M. Turco S.J. J. Biol. Chem. 1997; 272: 3799-3805Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) argues that the Vrg4 protein functions as a transporter per say, rather than as a regulator of transport. It is the concerted action of both the glycosyltransferases and the NSTs that ultimately dictate glycoconjugate synthesis. Although much is known about the glycosyltransferases, relatively little is known about the biochemical and molecular basis by which the NSTs transport nucleotide sugars from the cytosol into the Golgi. We have undertaken an analysis of the Vrg4 protein to gain a better understanding of NST function. Here data are presented that suggest this protein functions as a homodimer. In addition, deletion analysis of mutant proteins has enabled us to identify a region of the protein that is essential for its exit from the ER and proper Golgi localization but that, when mutated, does not interfere with protein stability or oligomerization. This N-terminal region is distinct from a C-terminal domain required for oligomerization (1.Abe M. Hashimoto H. Yoda K. FEBS Lett. 1999; 458: 309-312Crossref PubMed Scopus (38) Google Scholar) and protein stability. Like Vrg4 proteins lacking the cytosolic N-terminal domain, these Vrg4ΔCproteins are also retained in the ER (1.Abe M. Hashimoto H. Yoda K. FEBS Lett. 1999; 458: 309-312Crossref PubMed Scopus (38) Google Scholar) but probably because they are misfolded and incompletely assembled. When present on a protein that resides in the ER, a sequence homologous to the N terminus of Vrg4p targets the heterologous protein to the Golgi, suggesting that sequences in the N terminus act as a positive ER export signal. Standard yeast media and genetic techniques were used (11.Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 3-20Crossref PubMed Scopus (2545) Google Scholar). Hygromycin B sensitivity was tested on yeast extract/peptone/adenine sulfate/dextrose plates (YPAD) supplemented with 50 μg/ml hygromycin B (Roche Molecular Biochemicals) as described previously (12.Dean N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1287-1291Crossref PubMed Scopus (138) Google Scholar). SEY6210 (MATα ura3–52 leu2–3, 112 his3-Δ200 trp1-Δ901 lys2–801 suc2Δ9) was used as the parental strain for the construction of XGY10 and XGY12, in which the chromosomal alleles of VRG4 or GDA1, respectively, are replaced with those that contain a C-terminal tobacco etch virus (TEV) protease cleavage site fused to a protein A tag (see below). XGY11 was constructed from W303a (MATa ade2–1 ura3–1 his3–11 trp1–1 leu2–3, 112 can1–100) and contains a replacement of the normal VRG4 locus with an allele in whichVRG4 is tagged at the N terminus with a triple HA-TEV tag and is under the control of the GAL1 promoter. NDY5 (MATα ura3–52 leu2–211 vrg4–2) (9.Poster J.B. Dean N. J. Biol. Chem. 1996; 271: 3837-3845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) was used for complementation analysis of the vrg4–2 allele. XGY13 is a derivative of RSY255 (MATα ura3–52 leu2–211) but contains an HA-tagged allele of VRG4integrated at the ura3–52 locus, driven by theTPI promoter (8.Dean N. Zhang Y.B. Poster J.B. J. Biol. Chem. 1997; 272: 31908-31914Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The chromosomal VRG4 and GDA1 alleles were tagged at the 3′-end with sequences encoding the TEV protease cleavage site followed by the protein A IgG binding domain, using PCR-mediated gene modification by homologous recombination (13.Baudin A. Ozier-Kalogeropoulos O. Denouel A. Lacroute F. Cullin C. Nucleic Acids Res. 1993; 21: 3329-3330Crossref PubMed Scopus (1127) Google Scholar, 14.Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2241) Google Scholar, 15.Wach A. Brachat A. Alberti-Segui C. Rebischung C. Philippsen P. Yeast. 1997; 13: 1065-1075Crossref PubMed Scopus (508) Google Scholar). A fragment, encoding the TEV protease cleavage site (SENLYFQG) (16.Mondigler M. Ehrmann M. J. Bacteriol. 1996; 178: 2986-2988Crossref PubMed Google Scholar) followed by the protein A "Z" domains and the Schizosaccharomyces pombe HIS5gene, was amplified from the plasmid pZZ-HIS5 (17.Rayner J.C. Munro S. J. Biol. Chem. 1998; 273: 26836-26843Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) and targeted to the 3′-end of either the VRG4 or GDA1 gene. Similarly, the chromosomal VRG4 allele was tagged at the 5′-end with sequences encoding three copies of the HA epitope and the TEV protease cleavage site. A fragment was amplified by PCR from plasmid pFA6a-His3MX6-PGAL1–3HA (18.Longtine M.S. McKenzie III, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4193) Google Scholar), which contains theHIS5 marker and the GAL1 promoter followed by the HA tag. A TEV protease cleavage site was inserted at the junction between the first methionine of the VRG4 ORF and the HA tag by including DNA sequences encoding this cleavage site sequence directly in the reverse primer used in the PCR amplification. Plasmids used in this study are listed in TableI. Standard molecular biology techniques were used for all plasmid constructions (19.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar).Table IPlasmids used in this studyPlasmidDescriptionSourcepBluescript SK−Cloning vectorStratagenepSK−P/X HA3Cloning vector for COOH HA3 tagRef. 24.Neiman A.M. Mhaiskar V. Manus V. Galibert F. Dean N. Genetics. 1997; 145: 637-645Crossref PubMed Google ScholarpSK−P/X myc3Cloning vector for COOH Myc3 tagThis studypRS316URA3/CEN6 yeast shuttle vectorRef. 20.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google ScholarYEplac181LEU2/2μ yeast shuttle vectorRef. 21.Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2528) Google ScholarYEp352URA3/2μ yeast shuttle vectorRef. 22.Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1993; 2: 163-167Crossref Scopus (1083) Google ScholarpRHL-myc3VRG4-myc3/URA3/CEN6This studyYEplac181-RHL-myc3VRG4-myc3/LEU2/2μThis studyYEp352-RHL-myc3VRG4-myc3/URA3/2μThis studypRHL-HA3VRG4-HA3/URA3/CEN6Ref. 9.Poster J.B. Dean N. J. Biol. Chem. 1996; 271: 3837-3845Abstract Full Text Full Text PDF PubMed Scopus (62) Google ScholarYEplac181-RHL-HA3VRG4-HA3/LEU2/2μThis studyYEp352 RHL-HA3VRG4-HA3/URA3/2μThis studyYEpGAPTDH promoter in URA3/2μ yeast shuttle vectorRef. 23.Gao X.D. Kaigorodov V. Jigami Y. J. Biol. Chem. 1999; 274: 21450-21456Abstract Full Text Full Text PDF PubMed Scopus (81) Google ScholarYEpGAP-N-myc3Cloning vector forNH2 Myc3 tagThis studyYEpGAPVRG4-HA3TDH-VRG4-HA3/URA3/2μThis studyYEpGAPΔ15N-HA3TDH-vrg4-Δ15N-HA3/URA3/2μThis studyYEpGAPΔ44N-HA3TDH-vrg4-Δ44N-HA3/URA3/2μThis studyYEpGAPΔ78N-HA3TDH-vrg4-Δ78N-HA3/URA3/2μThis studyYEpGAP-Ypl244-HA3TDH-YPL244-HA3/URA3/2μThis studyYEpGAP-Ypl244Δ41-HA3TDH-YPL244Δ41-HA3/URA3/2μThis studyYEpGAP-VN47Ypl244Δ41-HA3TDH-VN47YPL244Δ413/URA3/2μThis studyYEplacVRG4-myc3TDH-VRG4-myc3/LEU2/2μThis studyYEplacΔ15N-myc3TDH-vrg4-Δ15N-myc3/LEU2/2μThis studyYEplacΔ44N-myc3TDH-vrg4-Δ44N-myc3/LEU2/2μThis studyYEplacΔ78N-myc3TDH-vrg4-Δ78N-myc3/LEU23/2μThis studyYEpGAPVRG4-N-myc3TDH-VRG4-myc3/URA3/2μThis studyYEpGAPΔ6C-myc3TDH-vrg4-Δ6C-myc3/URA3/2μThis studyYEpGAPΔ13C-myc3TDH-vrg4-Δ13C-myc3/URA3/2μThis studyYEpGAPΔ34C-myc3TDH-vrg4-Δ34C-myc3/URA3/2μThis studypRS TPI-HVG1-HA3TPI-HVG1-HA3/URA3/CEN6This studyYEpTPI-HVG1-HA3TPI-HVG1: HA 3/URA3/2μThis studypRS TPI-mHVG1-HA3TPI-mHVG1-HA3/URA3/CEN6This studyYEpTPI-mHVG1-HA3TPI-mHVG1: HA 3/URA3/2μThis study Open table in a new tab To construct pRHL-myc3, the VRG4 gene was cloned in-frame to sequences encoding three tandem copies of the myc epitope (EQKLISEEDL). A HindIII/NsiI fragment containing the VRG4 ORF lacking the stop codon was isolated by PCR and cloned into pSK−P/X myc3, a derivative of Bluescript SK− (Stratagene). pSK−P/X myc3 carries a 172-base pair fragment encoding three tandem copies of the myc epitope, cloned between the PstI andXbaI sites of pBluescript SK−. pSK−VRG4-myc3 encodes Vrg4p fused to three copies of the myc epitope at the C terminus. SK−VRG4-myc3 was used to generate pRHL-myc3, which contains VRG4-myc 3, under the control of its own promoter, on anEcoRI/HindIII fragment in theCEN6/URA3 vector, pRS316 (20.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). ThisEcoRI/HindIII fragment from pRHL-myc3was cloned into YEplac181 (21.Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2528) Google Scholar) to generate YEplac181-RHL-myc3 to allow expression in a 2μ/LEU2 vector and into YEp352 (22.Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1993; 2: 163-167Crossref Scopus (1083) Google Scholar) to generate YEp352-RHL-myc3 to allow expression in a 2μ/URA3 vector. Identical constructs, but containing the triple HA tag at the C terminus are pRHL-HA3, YEplac181-RHL-HA3, and YEp352 RHL-HA3. A series of plasmids containing vrg4 alleles with 5′ deletions were constructed by PCR amplification using pSK-RHL HA3 as the template plasmid (8.Dean N. Zhang Y.B. Poster J.B. J. Biol. Chem. 1997; 272: 31908-31914Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Each of the deletion constructs shares the same 3′-end, including sequences encoding the C-terminal triple HA-tag. KpnI/SmaI amplified fragments, amplified by PCR, containing deleted 5′ termini ofVRG4 were ligated into the KpnI/PvuII sites of pYEpGAP (23.Gao X.D. Kaigorodov V. Jigami Y. J. Biol. Chem. 1999; 274: 21450-21456Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) to place them under the control of the glyceraldehyde-3-phosphate dehydrogenase (TDH3) promoter in a 2μ/URA3 yeast expression plasmid. An ATG sequence in YEpGAP encodes the initiating methionine in each thesevrg4ΔN alleles. 5′ primers for these PCR reactions were designed to amplify from the codon encoding the 16th, 45th, or 79th amino acid of Vrg4p, respectively, to generate the series pYEpGAPΔ15N-HA3, pYEpGAPΔ44N-HA3, and pYEpGAPΔ78N-HA3. Similarly, anEcoRI/SmaI fragment containing the entire wild type VRG4-HA gene was amplified by PCR and ligated into theEcoRI/PvuII sites of YEp352GAP vector to create pYEpGAPVRG4-HA3. An analogous series of plasmids, encoding Vrg4ΔN proteins that were myc-tagged, was derived from the YEpGAPΔN plasmids described above by replacing the 3′-half of eachVRG4 gene with a HpaI/SacI fragment from pRHL-myc3. Each of the BamHI/SacI fragments were inserted into Yeplac181. This series of plasmids encode Vrg4p with N-terminal deletions (Δ15, Δ44, and Δ78) and a C-terminal myc epitope, whose expression is under the control of the TDH3promoter in a 2μ/LEU2 plasmid. To construct plasmids encoding C-terminal Vrg4p deletion mutants, we first made a vector (YEpGAP-N-myc3) containing a fragment encoding three copies of the myc epitope, flanked by anEcoRI/KpnI site and containing an initiating ATG, in YEpGAP. KpnI/SmaI DNA fragments containing deleted 3′ termini of VRG4 were amplified and ligated into the KpnI/PvuII sites of YEpGAP, in-frame and 3′ to the myc3 epitope. The 3′ PCR primers were designed to delete the last 6, 13, or 34 amino acids of Vrg4p to generate YEpGAPΔ6C-myc3, YEpGAPΔ13C-myc3, and YEpGAPΔ34C-myc3. These plasmids encodeVrg4p C-terminal deletions with an N-terminal myc epitope, whose expression is under the control of the TDH3 promoter in a 2μ/URA3plasmid. An HA-tagged allele of the HVG1 gene was created in several steps. First, a fragment containing the HVG1 ORF, lacking the stop codon and flanked by a 5′ HindIII and a 3′NsiI site was amplified by PCR from yeast genomic DNA and cloned into pSK−P/X HA3 (24.Neiman A.M. Mhaiskar V. Manus V. Galibert F. Dean N. Genetics. 1997; 145: 637-645Crossref PubMed Google Scholar) to generate pSK−HVG1-HA3. This plasmid encodes Hvg1p with the HA3 epitope at the C terminus. AHindIII/NotI fragment from SK−HVG1-HA3 containing HA-taggedHVG1 was cloned into pRS316TPI (9.Poster J.B. Dean N. J. Biol. Chem. 1996; 271: 3837-3845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) to generate pRS TPI-HVG1-HA3. This places HA-tagged HVG1 under the control of the TPI promoter in a CEN6/URA3yeast expression plasmid. YEpTPI-HVG1-HA3 contains theSalI/SacI fragment from pRS TPI-HVG1-HA3 in pYEp352. To construct a plasmid that encodes Hvg1p with an N-terminal extension homologous to that of Vrg4p, site-directed mutagenesis was used to change the stop codon located 60 base pairs upstream the first ATG of the HVG1 ORF to an arginine codon normally found at amino acid 78 of the Vrg4 protein. A HindIII/NsiI fragment containing the entire HVG1 ORF as well as 275 base pairs of 5′-flanking sequences was isolated by PCR and cloned into pSK−P/X HA3 to place the HA3epitope at the 3′-end. This plasmid was used as template DNA for site-directed mutagenesis of HVG1, using the QuikChangeTM Site-Directed Mutagenesis kit (Stratagene). The mutagenic primers change the T of the stop codon (TGA) to a C to produce an arginine codon (CGA). This generated a 98-amino acid extension in the N terminus of HVG1 ORF to create pSK− mHVG1-HA3. A 1.13-kilobase pairHindIII/NotI fragment from pSK−mHVG1-HA3 was cloned into pRS316TPI to make pRSTPI-mHVG1-HA3. This places the mutated HVG1(mHVG1) under the control of the TPI promoter in a CEN6/URA3 yeast expression plasmid. Similar to YEpTPI-HVG1-HA3, YEpTPI-mHVG1-HA3 was also produced by cloning the SalI/SacI fragment from pRS TPI-mHVG1-HA3 into YEp352. A fragment containing the entire YPL244C ORF was isolated by PCR from yeast genomic DNA and cloned into pSK−P/X HA3 to place the HA3 epitope at the 3′-end of Ypl244p. YEpGAPYpl244-HA3 and YEpGAPYpl244Δ41-HA3 were constructed by cloning aKpnI/SmaI fragment (amplified by PCR) into YEpGAP. YEpGAP-VN47Ypl244Δ41-HA3 was constructed by fusing a fragment encoding the N-terminal 47 amino acids of Vrg4p in-frame into the EcoRI/KpnI site of YEpGAPYpl244Δ41-HA3. Exponentially growing yeast cells (A 600: 1–3) were harvested and converted to spheroplasts with lyticase, as described previously (25.Chi J.H. Roos J. Dean N. J. Biol. Chem. 1996; 271: 3132-3140Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Spheroplasts from 3–4 OD units of cells were resuspended in 400 μl of ice-cold lysis buffer (150 mm NaCl, 10 mmHEPES-KOH (pH 7.5), 5 mm MgCl2, 1 mm PMSF) 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 for both FPLC analysis and the co-immunoprecipitation assays described below. For preparation of a membrane fraction, 50 A 600units of cells were spheroplasted using lyticase (25.Chi J.H. Roos J. Dean N. J. Biol. Chem. 1996; 271: 3132-3140Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The spheroplasts were resuspended in 1 ml of cold lysis buffer (0.1 msorbitol, 50 mm potassium acetate, 2 mm EDTA, 20 mm HEPES (pH 7.4), 1 mm dithiothreitol) containing a protease inhibitor mixture (1 mm PMSF, 1 μg/ml pepstatin, 50 μg/ml N-tosyl-l-lysine chloromethyl ketone, 100 μg/mlN-tosyl-l-phenylalanine chloromethyl ketone, and 100 μg/ml trypsin inhibitor). Lysis was carried out by Dounce homogenization (25 strokes) on ice, and unbroken cells were removed from the lysate by centrifugation for 5 min in a Microfuge. Membranes were isolated by centrifugation at 100,000 × g for 30 min in a Beckman Optima TL ultracentrifuge. The membrane pellet was resuspended in 150 μl of lysis buffer and used for protease protection assays (see below). The HA-tagged proteins were immunoprecipitated by incubating 400 μl of the detergent extract (described above) 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. Immunoprecipitation of myc-tagged proteins was done identically, except we used culture supernatants containing the 9E10 monoclonal ant-myc antibody and the incubations were carried out at 4 °C overnight. The protein A-Sepharose beads and associated proteins were centrifuged and washed three times with the same lysis buffer (1% digitonin or 1% Triton X-100, 150 mm NaCl, 50 mm HEPES-KOH (pH 7.5), 5 mm MgCl2, 1 mm PMSF). After resuspending in Laemmli's sample buffer and solubilizing at 45 °C for 3 min, immunoprecipitates were fractionated by 10% SDS-PAGE, transferred to Immobilon-polyvinylidene difluoride membranes (Millipore) and immunoblotted with anti-HA (Y-11) or anti-myc A-14 rabbit polyclonal antibodies (Santa Cruz Biotechnology). Secondary anti-rabbit antibodies conjugated to horseradish peroxidase (Amersham Pharmacia Biotech) were used at a 1:3000 dilution and detected by chemiluminescence (ECL, Amersham Pharmacia Biotech). Whole cell protein extracts were prepared by trichloroacetic acid precipitation, as described previously (25.Chi J.H. Roos J. Dean N. J. Biol. Chem. 1996; 271: 3132-3140Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Proteins were separated by SDS-PAGE and detected by Western immunoblotting using anti-HA or anti-myc antibodies, as described previously (9.Poster J.B. Dean N. J. Biol. Chem. 1996; 271: 3837-3845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Culture supernatants, containing the monoclonal anti-HA antibody, 12CA5, or the monoclonal anti-myc antibody, 9E10, were used at a 1:10 dilution. Rabbit polyclonal IgG against the HA epitope (Y-11) and c-myc epitope (A-14) (Santa Cruz) were used at a 1:2000 dilution. Rabbit anti-mouse IgGs (Jackson ImmunoResearch Laboratories), used for the detection of the protein A tag, were used at a 1:5000 dilution. Secondary anti-rabbit or anti-mouse antibodies (Amersham Pharmacia Biotech), conjugated to horseradish peroxidase, were used at a 1:3000 dilution and were detected by chemiluminescence (ECL, Amersham Pharmacia Biotech). Indirect immunofluorescence of yeast cells was performed as described previously (8.Dean N. Zhang Y.B. Poster J.B. J. Biol. Chem. 1997; 272: 31908-31914Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Samples were observed with a Zeiss Axioscop and photographed with a Sony DXC-9000 cooled CCD camera. Images were captured using NIH Image software, and all processing was done with Canvas (v.5) (Deneba). Membrane fractions (10 μl) were prepared as described above and were mixed with 20 units of TEV protease (Life Technologies, Inc.) in a reaction containing (100 mm sorbitol, 50 mm Tris-HCl (pH 8.0), 0.5 mm EDTA, 1 mm dithiothreitol) in a final volume of 60 μl. In control reactions, Triton X-100 or zinc sulfate was added to a final concentration of 0.5% or 0.1 m, respectively. Reactions were incubated for 1 h at 30 °C. After diluting in SDS loading buffer to stop the reaction, proteins were separated by SDS-PAGE and further analyzed for proteolysis by immunoblotting with Rabbit anti-mouse IgGs for the detection of the protein A epitope or 12CA5 for the detection of the HA epitope, as described above. 1% Triton X-100 or 1% digitonin cell-free extracts were prepared exactly as described above from yeast expressing the HA-tagged VRG4 allele (pRHL-HA3). 200 μl of extract were fractionated over a gel filtration column (Superose 6 HR 10/30, Amersham Pharmacia Biotech) equilibrated with 150 mm NaCl, 50 mm HEPES-KOH (pH 7.5), 5 mm MgCl2, 1 mm PMSF, containing either 1% Triton X-100 (for Triton extracts) or 1% digitonin (for digitonin extracts), using the FPLC system (Amersham Pharmacia Biotech). FPLC was performed at a flow rate of 0.2 ml/min, and 1-ml fractions were collected. Fractions were analyzed for the presence of Vrg4-HAp by SDS-PAGE, followed by immunoblotting with HA-specific antibodies. For determination of molecular weight, a calibration kit containing proteins that range from 29,000 to 700,000 (Amersham Pharmacia Biotech) was also fractionated by FPLC in parallel and detected by staining with Coomassie Blue. Gas1p, which was also used as a molecular weight marker, was detected by Western blotting using anti-Gas1p polyclonal antibodies. Using co-immunoprecipitation assays of detergent-solubilized, epitope-tagged Vrg4 proteins, coincident with other studies (1.Abe M. Hashimoto H. Yoda K. FEBS Lett. 1999; 458: 309-312Crossref PubMed Scopus (38) Google Scholar) we found that Vrg4p exists as a multimer. To investigate its oligomeric properties, the molecular weight of the Vrg4p-containing complex was examined by gel filtration chromatography. Detergent extracts prepared from yeast expressing an HA-tagged VRG4 allele were fractionated by FPLC over a Superose 6 column. This tagged allele can complement the hygromycin B sensitivity of a vrg4 mutant, indicating that this epitope does not alter the normal function of Vrg4p (8.Dean N. Zhang Y.B. Poster J.B. J. Biol. Chem. 1997; 272: 31908-31914Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Protein extracts were prepared under conditions that favored stable Vrg4p-containing oligomeric complexes, assayed by the co-immunoprecipitation of HA- and myc-tagged Vrg4 proteins (e.g. see Fig. 4 and "Experimental Procedures"). Either 1% Triton X-100 or 1% digitonin was used to solubilize proteins, and extracts were fractionated in the same lysis buffer, to maintain stable complexes throughout the procedure. Fractions were analyzed for the presence of Vrg4-HAp by SDS-PAGE, followed by immunoblotting with HA-specific antibodies. A comparison of the distribution of Vrg4-HAp and molecular weight standards showed that the peak position of Vrg4-HAp elution corresponds to a molecular mass of about 67 kDa (Fig.1), which is about twice the predicted molecular mass of monomeric Vrg4p. The peak of Vrg4p elution was the same whether the fractionation was performed with buffer containing 1% Triton X-100 or 1% digitonin (Fig. 1). This result suggested that the size of Vrg4p-containing complex is similar in the presence of both 1% Triton and 1% digitonin. The majority of Vrg4-HAp prepared from digitonin extracts was recovered in a sharp, symmetrical peak. No material of a lower molecular weight could be detected, suggesting that most of the Vrg4p extracted from cells by digitonin exists as a homogenous higher molecular weight species. In contrast, when prepared from Triton extracts, Vrg4p was recovered in a broader peak, with a trailing edge of fractions containing monomeric forms of Vrg4 that co-eluted with lower molecular weight standards. This observation is consistent with our results tha
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