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• W2012243695 abstract "Yeast Ypt6p, the homologue of the mammalian Rab6 GTPase, is not essential for cell viability. Based on previous studies with ypt6 deletion mutants, a regulatory role of the GTPase either in protein retrieval to the trans-Golgi network or in forward transport between the endoplasmic reticulum (ER) and early Golgi compartments was proposed. To assess better the primary role(s) of Ypt6p, temperature-sensitive ypt6 mutants were generated and analyzed biochemically and genetically. Defects inN-glycosylation of proteins passing the Golgi and of Golgi-resident glycosyltransferases as well as protein sorting defects in the trans-Golgi were recorded shortly after functional loss of Ypt6p. ER-to-Golgi transport and protein secretion were delayed but not interrupted. Mis-sorting of the vesicular SNARE Sec22p to the late Golgi was also observed. Combination of theypt6-2 mutant allele with a number of mutants in forward and retrograde transport between ER, Golgi, and endosomes led to synthetic negative growth defects. The results obtained indicate that Ypt6p acts in endosome-to-Golgi, in intra-Golgi retrograde transport, and possibly also in Golgi-to-ER trafficking. Yeast Ypt6p, the homologue of the mammalian Rab6 GTPase, is not essential for cell viability. Based on previous studies with ypt6 deletion mutants, a regulatory role of the GTPase either in protein retrieval to the trans-Golgi network or in forward transport between the endoplasmic reticulum (ER) and early Golgi compartments was proposed. To assess better the primary role(s) of Ypt6p, temperature-sensitive ypt6 mutants were generated and analyzed biochemically and genetically. Defects inN-glycosylation of proteins passing the Golgi and of Golgi-resident glycosyltransferases as well as protein sorting defects in the trans-Golgi were recorded shortly after functional loss of Ypt6p. ER-to-Golgi transport and protein secretion were delayed but not interrupted. Mis-sorting of the vesicular SNARE Sec22p to the late Golgi was also observed. Combination of theypt6-2 mutant allele with a number of mutants in forward and retrograde transport between ER, Golgi, and endosomes led to synthetic negative growth defects. The results obtained indicate that Ypt6p acts in endosome-to-Golgi, in intra-Golgi retrograde transport, and possibly also in Golgi-to-ER trafficking. endoplasmic reticulum trans-Golgi network open reading frame peptide: N-glycosidase coat protein soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor wild type alkaline phosphatase Intercompartmental transport of proteins in secretion and endocytosis is affected by a variety of vesicle populations, each having its own biochemical identity. Generation of transport vesicles at different donor compartments and their fusion with defined target membranes follow general principles and require protein machines made up of related and evolutionarily conserved proteins that are, however, specific for each transport step (1Jahn R. Sudhof T.C. Annu. Rev. Biochem. 1999; 68: 863-911Crossref PubMed Scopus (1020) Google Scholar, 2Mellman I. Warren G. Cell. 2000; 100: 99-112Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). Among the proteins that confer specificity and directionality to vesicular trafficking are Ras-like GTPases of the Ypt/Rab family (3Lazar T. Gotte M. Gallwitz D. Trends Biochem. Sci. 1997; 22: 468-472Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 4Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 5Segev N. Curr. Opin. Cell Biol. 2001; 13: 500-511Crossref PubMed Scopus (247) Google Scholar, 6Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2700) Google Scholar). In the yeast Saccharomyces cerevisiae, this GTPase family has 11 members of which only those that act in forward transport through the secretory pathway (Ypt1p, Sec4p, and the pair of apparently redundant Ypt31/Ypt32-GTPases) are essential for cell viability (3Lazar T. Gotte M. Gallwitz D. Trends Biochem. Sci. 1997; 22: 468-472Abstract Full Text PDF PubMed Scopus (187) Google Scholar). Of the nonessential ones, Ypt6p, the homologue of the mammalian Rab6 GTPases, has been implicated in having a role in the retrieval of proteins from endosomes to thetrans-Golgi network (7Tsukada M. Gallwitz D. J. Cell Sci. 1996; 109: 2471-2481PubMed Google Scholar, 8Tsukada M. Will E. Gallwitz D. Mol. Biol. Cell. 1999; 10: 63-75Crossref PubMed Scopus (77) Google Scholar, 9Bensen E.S. Yeung B.G. Payne G.S. Mol. Biol. Cell. 2001; 12: 13-26Crossref PubMed Scopus (62) Google Scholar, 10Siniossoglou S. Pelham H.R. EMBO J. 2001; 20: 5991-5998Crossref PubMed Scopus (162) Google Scholar) or in anterograde (11Li B. Warner J.R. J. Biol. Chem. 1996; 271: 16813-16819Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Li B. Warner J.R. Yeast. 1998; 14: 915-922Crossref PubMed Scopus (22) Google Scholar) and retrograde (9Bensen E.S. Yeung B.G. Payne G.S. Mol. Biol. Cell. 2001; 12: 13-26Crossref PubMed Scopus (62) Google Scholar) Golgi transport. The divergent views on Ypt6p function were based on the results of the initial studies (7Tsukada M. Gallwitz D. J. Cell Sci. 1996; 109: 2471-2481PubMed Google Scholar, 8Tsukada M. Will E. Gallwitz D. Mol. Biol. Cell. 1999; 10: 63-75Crossref PubMed Scopus (77) Google Scholar, 11Li B. Warner J.R. J. Biol. Chem. 1996; 271: 16813-16819Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Li B. Warner J.R. Yeast. 1998; 14: 915-922Crossref PubMed Scopus (22) Google Scholar) using ypt6 deletion or truncation mutants. Such mutants were found to be growth-inhibited at elevated temperature, to partially mis-sort proteins in thetrans-Golgi network already at permissive temperature, and to moderately interfere with protein secretion at nonpermissive conditions (7Tsukada M. Gallwitz D. J. Cell Sci. 1996; 109: 2471-2481PubMed Google Scholar, 11Li B. Warner J.R. J. Biol. Chem. 1996; 271: 16813-16819Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Temperature sensitivity of ypt6 deletion mutants allowed us to isolate multicopy suppressors of which one,SYS1 (7Tsukada M. Gallwitz D. J. Cell Sci. 1996; 109: 2471-2481PubMed Google Scholar), could also rescue another protein mis-sorting mutant, ricΔ (9Bensen E.S. Yeung B.G. Payne G.S. Mol. Biol. Cell. 2001; 12: 13-26Crossref PubMed Scopus (62) Google Scholar), later shown to be defective in the nucleotide exchange factor for Ypt6p (13Siniossoglou S. Peak-Chew S.Y. Pelham H.R. EMBO J. 2000; 19: 4885-4894Crossref PubMed Scopus (138) Google Scholar). Cells lacking Ypt6p accumulate transport vesicles (8Tsukada M. Will E. Gallwitz D. Mol. Biol. Cell. 1999; 10: 63-75Crossref PubMed Scopus (77) Google Scholar) of which at least a fraction may be derived from endosomes and destined to fuse with late Golgi membranes (10Siniossoglou S. Pelham H.R. EMBO J. 2001; 20: 5991-5998Crossref PubMed Scopus (162) Google Scholar). On the other hand, temperature-sensitive growth ofypt6 deletion mutants can be overcome efficiently by raising the intracellular level of Ypt1p (12Li B. Warner J.R. Yeast. 1998; 14: 915-922Crossref PubMed Scopus (22) Google Scholar), the GTPase required for ER1-to-Golgi (14Cao X. Ballew N. Barlowe C. EMBO J. 1998; 17: 2156-2165Crossref PubMed Scopus (291) Google Scholar) and early Golgi transport (15Jedd G. Richardson C. Litt R. Segev N. J. Cell Biol. 1995; 131: 583-590Crossref PubMed Scopus (128) Google Scholar). Furthermore, partial suppression of growth defects of ypt6 and ric1 null mutants by high expression of a variety of genes that are known to act in either forward or retrograde Golgi transport have led to the assumption that Ypt6p might participate in regulating more than one transport step (9Bensen E.S. Yeung B.G. Payne G.S. Mol. Biol. Cell. 2001; 12: 13-26Crossref PubMed Scopus (62) Google Scholar). This would in fact mirror the situation in mammalian cells where the homologue of yeast Ypt6p, Rab6, initially thought to function only inintra-Golgi transport (16Martinez O. Schmidt A. Salamero J. Hoflack B. Roa M. Goud B. J. Cell Biol. 1994; 127: 1575-1588Crossref PubMed Scopus (221) Google Scholar), is also required for recycling of proteins between endosomes and the TGN (17Mallard F. Tang B.L. Galli T. Tenza D. Saint-Pol A. Yue X. Antony C. Hong W. Goud B. Johannes L. J. Cell Biol. 2002; 156: 653-664Crossref PubMed Scopus (421) Google Scholar). The notable difference, however, is that although yeast has only one form of Ypt6p, mammalian cells are endowed with two Rab6 isoforms that apparently act sequentially in transport from endosomes to the early Golgi (17Mallard F. Tang B.L. Galli T. Tenza D. Saint-Pol A. Yue X. Antony C. Hong W. Goud B. Johannes L. J. Cell Biol. 2002; 156: 653-664Crossref PubMed Scopus (421) Google Scholar) and even to the ER (18White J. Johannes L. Mallard F. Girod A. Grill S. Reinsch S. Keller P. Tzschaschel B. Echard A. Goud B. Stelzer E.H. J. Cell Biol. 1999; 147: 743-760Crossref PubMed Scopus (363) Google Scholar). As previous studies with ypt6 deletion mutants have the inherent problem that adaptive changes in the physiology of such cells might obscure the true lesions caused by functional loss of the GTPase, we sought to investigate Ypt6p function with the help of conditional mutants. The data obtained from genetic interactions and from experiments following the kinetics of protein transport and sorting as well as the state of protein glycosylation at permissive and nonpermissive conditions are best explained by Ypt6p acting both in recycling of proteins from endosomes to the Golgi and from late to early Golgi compartments. Yeast strains used in this study are listed in Table I. Yeast transformations, mating, sporulation, and tetrad analyses were performed using standard techniques (19Gietz D., St Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2887) Google Scholar, 20Sherman F. Hicks J. Methods Enzymol. 1991; 194: 21-37Crossref PubMed Scopus (207) Google Scholar). c-Myc epitope tagging of ORFs was performed as described previously (21De Antoni A. Gallwitz D. Gene (Amst.). 2000; 246: 179-185Crossref PubMed Scopus (86) Google Scholar). To produce pKS-YPT6-URA3 for site-directed mutagenesis, the 1.4-kb XbaIYPT6 gene fragment from pRS315-YPT6 was inserted into theXbaI site of pBluescript II KS+ (Stratagene). The HindIII URA3 gene fragment from YEp24 (New England Biolabs) was inserted into the StuI site ofYPT6 created 22 bp behind the stop codon. To create pYX242-SEC35, the coding sequence of SEC35 gene with a newly created NcoI site at the 5′ end and an XhoI site at the 3′ end was amplified by PCR from yeast genomic DNA usingPfu DNA polymerase (Stratagene). This fragment was inserted into the NcoI-XhoI sites of pYX242 (R&D Systems). Plasmids pRS315-YPT6 and pRS325-SYS1 were constructed as described previously (7Tsukada M. Gallwitz D. J. Cell Sci. 1996; 109: 2471-2481PubMed Google Scholar); pRS326-YPT1 was from R. Peng (this laboratory), and pWB-Acycα (P CYC1-SEC22-myc-α factor,CEN, URA3) was from H. D. Schmitt (this laboratory).Table IStrains used in this studyStrainGenotypeSourceMSUC-1AMATa ura3 his3 leu2 trp1 ade8This laboratoryMSUC-3DMATα ura3 his3 leu2 trp1 lys2This laboratoryZLY1MSUC-3D,ypt6-1-URA3This studyZLY2MSUC-3D,ypt6-2-URA3This studyZLY2-KMSUC-3D,ypt6-2-loxP-KanMX-loxPThis studyZLY2-1AMSUC-1A,ypt6-2-URA3This studyZLY3MSUC-3D,ypt6-3-URA3This studyZLY4MSUC-3D,ypt6::HIS3This studyZLY45MSUC-3D,tlg2::HIS3This studyZLY361MSUC-3D,vps35::HIS3This studyZLY351MSUC-3D,OCH1-6His-2Myc-loxP-KanMX-loxPThis studyZLY352MSUC-3D,MNN1-6His-2Myc-loxP-KanMX-loxPThis studyZLY353MSUC-3D,MNN9-6His-2Myc-loxP-KanMX-loxPThis studyZLY354MSUC-3D,ANP1-6His-2Myc-loxP-KanMX-loxPThis studyZLY355MSUC-3D,VAN1-6His-2Myc-loxP-KanMX-loxPThis studyZLY356MSUC-3D, ypt6-2-URA3, OCH1-6His-2MYC-loxP-KanMX-loxPThis studyZLY357MSUC-3D, ypt6-2-URA3, MNN1-6His-2MYC-loxP-KanMX-loxPThis studyZLY358MSUC-3D, ypt6-2-URA3, MNN9-6His-2MYC-loxP-KanMX-loxPThis studyZLY359MSUC-3D, ypt6-2-URA3, ANP1-6His-2MYC-loxP-KanMX-loxPThis studyZLY360MSUC-3D, ypt6-2-URA3, VAN1-6His-2MYC-loxP-KanMX-loxPThis studyZLY184MATα ura3 his4 leu2 sec23-1 OCH1-6His-2MYC-loxP-KanMX-loxPThis studyZLY185MATα ura3 his4 leu2 sec23-1 MNN1-6His-2Myc-loxP-KanMX-loxPThis studyRPY18MSUC-1A,sec24-11Peng et al. (55Peng R., De Antoni A. Gallwitz D. J. Biol. Chem. 2000; 275: 11521-11528Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar)RPY41MSUC-3D,sed5-1This laboratoryRPY108MSUC-3D,sly1tsThis laboratoryRPY116MSUC,MATa ura3 his3 leu2 trp1 lys2 sec35-1This laboratoryYXY12αMSUC-3D, yip1-2-TRP1Yang et al. (40Yang X. Matern H.T. Gallwitz D. EMBO J. 1998; 17: 4954-4963Crossref PubMed Scopus (112) Google Scholar)YXY136MSUC-3D,ypt1 A136D -LEU2This laboratoryYLX15MSUC-3D, ypt32 A141D -HIS3 ypt31::KanMXThis laboratoryBSH-1BMATa ura3 his3 leu2 suc2-Δ9 bet1-1This laboratoryCTY1-1AMATaura3 his3 lys2 sec14-1S.D. Emr (UC San Diego)GWY67MATα leu2-3 112 ura3-52 trp1 uso1-1G. Waters (Princeton University)MB7MATα leu2 ura3 his4 suc2-Δ9 sec7-1M. Bielefeld (Universitat Düsseldorf)YTX50MATa ura3 leu2 his4 sec18-1T. Sommer (Max-Delbruck-Centrum Berlin)PC70MATα ura3 leu2 trp1 ret1-1P. Cosson (University of Geneva)PC130MATa ura3 leu2 his4 lys2 ret2-1P. CossonPC159MATa ura3 leu2 his4 trp1 suc2-Δ9 ret3-1P. CossonRH227-3AMATa ura3 his4 leu2 sec23-1H. Riazman (Biocenter, Basel)RH236-3AMATα ura3 his4 leu2 sec20-1H. RiezmanRH237-1AMATa ura3 his4 leu2 lys2 sec12-4H. RiezmanRH239-5AMATα ura3 his4 leu2 lys2 sec21-1H. RiezmanS27P4-9CMATα leu2 ura3 lys2 pep4::HIS3 sec27-1This laboratory Open table in a new tab YPT6 mutant genes encoding the GTPase with either K125N, G139D, or A143D substitution were generated by site-directed mutagenesis using QuickChangeTM mutagenesis kit (Stratagene) on the plasmid pKS-YPT6-URA3. After mutation, anXbaI fragment of the mutated YPT6 gene with the adjacent URA3 gene as selection marker was integrated into the YPT6 locus of a haploid strain, and mutants were selected on SD-Ura plates. Chromosomal mutations were verified by sequencing the PCR products generated from genomic DNA of selectedypt6 mutants. Disruption of genes was performed by PCR-based replacement using the HIS3 gene amplified from plasmid pRS303 (22Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to replace the respective ORFs as described (23Baudin A. Ozier-Kalogeropoulos O. Denouel A. Lacroute F. Cullin C. Nucleic Acids Res. 1993; 21: 3329-3330Crossref PubMed Scopus (1118) Google Scholar). Yeast cells were grown at 25 °C to mid-log phase. Ten A 600 units of cells were collected before and after incubation at 37 or 39 °C for different times, resuspended in 150 μl of SDS sample buffer containing proteinase inhibitors, and lysed by vortexing for 10 min at 4 °C with glass beads. For PNGase F digestion, cells were collected, spheroplasted, and gently lysed in HEPES buffer. The lysates were then centrifuged at 100,000 × g for 2 h at 4 °C; the pellets were resolved in 0.5% SDS buffer containing 1% β-mercaptoethanol, and digestions were done according to the protocols advertised by the supplier (New England Biolabs). For immunoblotting, proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with polyclonal antibodies against c-Myc epitope (Santa Cruz Biotechnology), CPY, ALP, or chitinase. Cell labeling and immunoprecipitations were performed as described previously (24Gaynor E.C. Emr S.D. J. Cell Biol. 1997; 136: 789-802Crossref PubMed Scopus (164) Google Scholar) with the following modifications. Cells were grown in synthetic minimal medium containing 2% glucose (SMM) (25Reid G.A. Methods Enzymol. 1983; 97: 324-329Crossref PubMed Scopus (19) Google Scholar) to mid-log phase and labeled with Tran35S-label (ICN) in either SD medium supplemented with the required amino acids or in SMM containing 1 m sorbitol and 1 mg/ml ovalbumin. Labeling in SMM was terminated by adding 10 mm NaN3/NaF and in SD medium by 2× spheroplast buffer (50 mm Tris, pH 7.5, 2 m sorbitol, 20 mm NaN3/NaF, 20 mm dithiothreitol). Spheroplasts were made by adding 10 μg of zymolase 100-T (Seikagaku Kogyo) per 1A 600 unit of cells and incubating for 45 min at 30 °C. Secretion of proteins into the medium was assayed as described (26Peyroche A. Jackson C.L. Methods Enzymol. 2001; 329: 290-300Crossref PubMed Scopus (14) Google Scholar). Invertase activity staining was carried out as described previously (27Benli M. Doring F. Robinson D.G. Yang X. Gallwitz D. EMBO J. 1996; 15: 6460-6475Crossref PubMed Scopus (139) Google Scholar). Yeast cells were grown at 25 °C to mid-log phase and then shifted to 37 °C for 1 h. 150 A 600 units of cells were collected. Lysates were prepared and subjected to sucrose gradient centrifugation as described (28Schröder S. Schimmoller F. Singer-Kruger B. Riezman H. J. Cell Biol. 1995; 131: 895-912Crossref PubMed Scopus (157) Google Scholar). Thirteen fractions were collected manually from the top of the gradient and mixed with one-fifth volume of 6× SDS sample buffer and incubated at 95 °C for 10 min prior to SDS-PAGE. Immunoblots were performed using specific antibodies against Kar2p, Emp47p, CPY, ALP, Anp1p, and the c-Myc epitope. Ypt6p is not required to sustain cell growth and multiplication. Therefore, previous studies (7Tsukada M. Gallwitz D. J. Cell Sci. 1996; 109: 2471-2481PubMed Google Scholar, 8Tsukada M. Will E. Gallwitz D. Mol. Biol. Cell. 1999; 10: 63-75Crossref PubMed Scopus (77) Google Scholar, 11Li B. Warner J.R. J. Biol. Chem. 1996; 271: 16813-16819Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Li B. Warner J.R. Yeast. 1998; 14: 915-922Crossref PubMed Scopus (22) Google Scholar) on the functional role of Ypt6p were performed with ypt6 deletion or truncation mutants which are, however, sensitive to growth at elevated temperature. Since mutants lacking a given protein are sometimes able to adapt to such a situation by activating a by-path for example, we sought to investigate Ypt6p function in conditional mutants. By analogy with other heat-sensitive Ypt proteins, three single amino acid substitutions were introduced into the conserved (NKXD) sequence, known to be involved in nucleotide binding, and into the helical region following this sequence (Fig. 1 A). Each of the three mutant alleles, ypt6-1 (expressing Ypt6(K125N)p), ypt6-2 (expressing Ypt6(G139D)p), andypt6-3 (expressing Ypt6(A143D)p), conferred heat sensitivity to a varying extent on haploid cells grown in rich media (Fig. 1 B). ypt6-2 cells exhibited the most severe growth defect. Like ypt6Δ cells of the same genetic background, this mutant grew slowly at 35 °C and failed to proliferate at 37 °C. The ypt6-1 mutant grew well at 37 °C but extremely slowly at 39 °C, whereas growth ofypt6-3 cells slowed down at 37 °C and completely failed at 39 °C. Compared with wild type, all ypt6 mutants exhibited somewhat reduced growth at 14 °C. The growth defects of ypt6 mutants at high temperature were partially rescued by the addition of 1 m sorbitol, a phenotype described for mutants with a defective cell wall (29Cid V.J. Duran A. del Rey F. Snyder M.P. Nombela C. Sanchez M. Microbiol. Rev. 1995; 59: 345-386Crossref PubMed Google Scholar). Therefore, we tested whether ypt6 mutants are sensitive to SDS and calcofluor white (CFW), agents affecting cell wall integrity and being more toxic to mutants with a defective cell wall. Compared with the wild type strain, ypt6-2 and ypt6 null mutants were more sensitive to 0.005% SDS and 0.005% CFW (Fig. 1 C), indicating a cell wall defect in these mutants. We selected vacuolar carboxypeptidase Y (CPY), alkaline phosphatase (ALP), and the secreted invertase as markers to test whether the intracellular vesicle transport was affected in the ypt6 mutants. These three proteins are N-linked glycoproteins that undergo core glycosylation in the ER and outer chain elongation in the Golgi before being transported to their final destination via different routes. CPY is transported via endosomes to the lumen of the vacuole, where it is processed to the mature form by proteolysis (30Stevens T. Esmon B. Schekman R. Cell. 1982; 30: 439-448Abstract Full Text PDF PubMed Scopus (371) Google Scholar). ALP is transported directly to the membrane of the vacuole without passing through endosomes (31Piper R.C. Bryant N.J. Stevens T.H. J. Cell Biol. 1997; 138: 531-545Crossref PubMed Scopus (134) Google Scholar) and is also cleaved by proteolysis in the vacuole. Invertase is rapidly transported to the periplasmic space (32Esmon B. Novick P. Schekman R. Cell. 1981; 25: 451-460Abstract Full Text PDF PubMed Scopus (169) Google Scholar). By monitoring the molecular size and/or localization, it can be determined which step of transport is affected in the mutants. At steady state, accumulation of core-glycosylated CPY (p1-form) was detected inypt6-1, ypt6-2, and ypt6-3 when shifted to the nonpermissive temperature (37 or 39 °C) for 1 and 2 h (Fig. 2 A). Further kinetic analysis of CPY by pulse-chase experiments showed that at permissive temperature (25 or 30 °C), ypt6 mutants exhibited somewhat delayed maturation of the enzyme and a partial mis-sorting of the Golgi-glycosylated p2 form to the extracellular space. At nonpermissive temperature, ypt6 mutants exhibited significantly reduced CPY transport kinetics. However, defects in the Golgi-specific glycosylation were demonstrated by the presence of a smear between p2CPY and p1CPY (compare wild type and mutants in Fig. 2, Band C). This is best seen in the experiment shown in Fig. 2 C in which cells initially grown at 25 °C were taken up in SMM containing 1 m sorbitol for cell stabilization, preincubated at either 25 or 37 °C for 10 to 60 min, and then radioactively labeled for 10 min at the respective temperature. Although the extent of CPY labeling decreased with the length of time the cells were incubated in the hypertonic medium, mis-sorting of a significant fraction of the Golgi-glycosylated vacuolar enzyme was evident already 10 min after shift of ypt6-2 cells to the nonpermissive temperature, and the p2 form of CPY decreased in size at later time points. At 60 min of preincubation at 37 °C, p2-CPY of the ypt6-2 mutant cells, in contrast to wild type cells, was detectable only as a smear above the sharp band of the ER core-glycosylated enzyme, clearly demonstrating a severe glycosylation defect in the Golgi. The defects observed in ypt6-2 mutants were more severe than those in ypt6-1 and ypt6-3cells, consistent with the growth phenotype. As shown in Fig. 3 A, pro-ALP was also accumulated in ypt6 mutants at steady state upon shift to nonpermissive temperature. Further analysis by pulse-chase experiments indicated that at 25 °C, maturation of ALP was normal inypt6-2 mutant cells. However, at 37 °C, pro-ALP was accumulated and, in gels, migrated progressively slower at chase times from 0 to 30 min (Fig. 3 B), suggesting ongoing glycosylation in the Golgi.Figure 3ALP maturation in ypt6mutants. A, Western blot analysis of ALP with total cellular protein. The same extracts used in the experiment of Fig. 2 A were analyzed with anti-ALP antibody. B, pulse-chase analysis with WT (MSUC-3D) and ypt6-2 (ZLY2) mutants. Cells were grown and subjected to pulse-chase analysis as described in the legend to Fig. 2 B. ALP was immunoprecipitated from cell lysates, resolved by SDS-PAGE, and visualized by autoradiography. * denotes an unrelated protein band that does not change electrophoretic behavior after PNGase F treatment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Invertase was analyzed both in the intracellular and extracellular fraction by activity staining in non-denaturing polyacrylamide gels. At permissive temperature, the secretion of highly glycosylated invertase was normal in ypt6 mutants as compared with wild type cells. At nonpermissive temperature, ypt6-1 and ypt6-3mutants showed slight defects in the secretion and glycosylation of the enzyme. Importantly, at 37 °C invertase in ypt6-2 mutant cells was clearly underglycosylated, and somewhat less than half of the enzyme was accumulated inside the cell. As controls for ER-to-Golgi transport mutants, processing and secretion of invertase was investigated in sec18-1 and ypt1 A136D strains at nonpermissive conditions. In these mutants, the enzyme was nearly completely trapped inside the cells in ER-glycosylated forms (Fig. 4 A). To analyze further whether Ypt6p is involved in secretion, we assayed for the export of proteins into the growth medium (24Gaynor E.C. Emr S.D. J. Cell Biol. 1997; 136: 789-802Crossref PubMed Scopus (164) Google Scholar, 26Peyroche A. Jackson C.L. Methods Enzymol. 2001; 329: 290-300Crossref PubMed Scopus (14) Google Scholar). Wild type and mutant cells were preincubated at 37 °C for 30 min and then subjected to pulse-chase at 37 °C. Cells were removed by centrifugation, and the proteins in the medium were precipitated with trichloroacetic acid, resolved by SDS-PAGE, and identified by autoradiography. As shown in Fig. 4 B, no protein band was detected in the medium ofsec18-1 cells, and only very faint bands were seen in the medium of ypt1 A136D cells, indicating the known secretion block in these two mutants. However, the same set of secreted proteins, including the most prominent band at 130–150 kDa that represents HSP150 (24Gaynor E.C. Emr S.D. J. Cell Biol. 1997; 136: 789-802Crossref PubMed Scopus (164) Google Scholar, 26Peyroche A. Jackson C.L. Methods Enzymol. 2001; 329: 290-300Crossref PubMed Scopus (14) Google Scholar), was visible in the medium of wild type,ypt6-2, and ret2-1 cells, the latter being defective in retrograde transport from the Golgi to the ER (33Cosson P. Demolliere C. Hennecke S. Duden R. Letourneur F. EMBO J. 1996; 15: 1792-1798Crossref PubMed Scopus (124) Google Scholar). Comparing the band intensities, it appears that secretion inypt6-2 mutants was somewhat reduced. We next examined the recycling of the v-SNARE Sec22p inypt6-2 mutant cells. Sec22p is known to cycle between the Golgi and the ER as well as between Golgi compartments. In wild type cells, only a small fraction of the v-SNARE reaches the late Golgi, but in mutants defective in retrograde transport of ER resident proteins, Sec22p is easily mis-sorted to this compartment (34Ballensiefen W. Ossipov D. Schmitt H.D. J. Cell Sci. 1998; 111: 1507-1520Crossref PubMed Google Scholar, 35Andag U. Neumann T. Schmitt H.D. J. Biol. Chem. 2001; 276: 39150-39160Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) in which the Kex2 protease resides. For this study, we took advantage of an Sec22-α factor fusion protein with a Kex2p cleavage site in its linker region and a c-Myc epitope tag adjacent to Sec22p. Cleavage of the fusion protein signals arrival in the late Golgi (34Ballensiefen W. Ossipov D. Schmitt H.D. J. Cell Sci. 1998; 111: 1507-1520Crossref PubMed Google Scholar). As shown in Fig. 4 C, the fraction of the fusion protein cleaved was significantly higher in ypt6-2 mutant cells compared with wild type cells independent of the temperature at which the experiment was conducted. This clearly indicates a recycling defect in Ypt6p-defective cells that could involve retrograde Golgi and/or Golgi-to-ER trafficking. The glycosylation defects of CPY and invertase inypt6 mutants prompted us to examine whether the defects resulted from instability of glycosyltransferases. In yeast cells, outer chain elongation of glycoproteins is catalyzed by glycosyltransferases in the Golgi. Among them, Anp1p, Mnn9p, and Van1p have been reported to recycle between the ER and the Golgi and are subject to mislocalization and to degradation in the vacuole when retrograde transport to the ER is blocked (36Todorow Z. Spang A. Carmack E. Yates J. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13643-13648Crossref PubMed Scopus (43) Google Scholar). We integrated c-Myc epitope-tagged versions of ANP1, MNN9, and VAN1into the genome of wild type and ypt6-2 strains and followed the fate of the tagged proteins by immunoblot analysis. As shown in Fig. 5 A, a shift of cells to 37 °C for 2 h did not result in a significant reduction of Anp1p-myc, Mnn9p-myc, and Van1p-myc in ypt6-2 compared with wild type cells. Also no redistribution of Mnn9p-myc and Anp1p was detected in ypt6-2 mutant cells in sucrose gradient analysis (Fig. 6 D), suggesting other reason(s) for the glycosylation defects observed. Likewise, there was no decrease of the Golgi protein Emp47p following shift ofypt6-2 cells to nonpermissive temperature (Fig. 5 A).Figure 6Distribution of proteins in sucrose gradients of ypt6-2 lysates. WT (A),ypt6-2 (B), and sec23-1 (C) strains expressing Myc-tagged Och1p (ZLY351, ZLY356, and ZLY184) were grown at 25 °C to mid-log phase and shifted to 37 °C for 1 h. Spheroplasts were generated and lysed gently. Lysates were fractionated by sucrose gradient (18–60%) centrifugation and assayed for the distribution of Och1p-myc, Emp47p, Kar2p, CPY, and ALP by immunoblot analysis using specific antibodies. Relative levels of proteins on immunoblots were quantified using a Lumi-Imager.D, WT and ypt6-2 strains expressing Mnn9p-myc (ZLY353 and ZLY358) were used to make lysates and subjected to fractionation and immunoblot analysis as described above.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Two o" @default.
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• W2012243695 title "Biochemical and Genetic Evidence for the Involvement of Yeast Ypt6-GTPase in Protein Retrieval to Different Golgi Compartments" @default.
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