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Vacuolar Localization of Oligomeric α-Mannosidase Requires the Cytoplasm to Vacuole Targeting and Autophagy Pathway Components in Saccharomyces cerevisiae

2001; Elsevier BV; Volume: 276; Issue: 23 Linguagem: Inglês

10.1074/jbc.m101150200

1083-351X

Maria U. Hutchins, Daniel J. Klionsky,

Fungal and yeast genetics research

One challenge facing eukaryotic cells is the post-translational import of proteins into organelles. This problem is exacerbated when the proteins assemble into large complexes. Aminopeptidase I (API) is a resident hydrolase of the vacuole/lysosome in the yeast Saccharomyces cerevisiae. The precursor form of API assembles into a dodecamer in the cytosol and maintains this oligomeric form during the import process. Vacuolar delivery of the precursor form of API requires a vesicular mechanism termed the cytoplasm to vacuole targeting (Cvt) pathway. Many components of the Cvt pathway are also used in the degradative autophagy pathway. α-Mannosidase (Ams1) is another resident hydrolase that enters the vacuole independent of the secretory pathway; however, its mechanism of vacuolar delivery has not been established. We show vacuolar localization of Ams1 is blocked in mutants that are defective in the Cvt and autophagy pathways. We have found that Ams1 forms an oligomer in the cytoplasm. The oligomeric form of Ams1 is also detected in subvacuolar vesicles in strains that are blocked in vesicle breakdown, indicating that it retains its oligomeric form during the import process. These results identify Ams1 as a second biosynthetic cargo protein of the Cvt and autophagy pathways. One challenge facing eukaryotic cells is the post-translational import of proteins into organelles. This problem is exacerbated when the proteins assemble into large complexes. Aminopeptidase I (API) is a resident hydrolase of the vacuole/lysosome in the yeast Saccharomyces cerevisiae. The precursor form of API assembles into a dodecamer in the cytosol and maintains this oligomeric form during the import process. Vacuolar delivery of the precursor form of API requires a vesicular mechanism termed the cytoplasm to vacuole targeting (Cvt) pathway. Many components of the Cvt pathway are also used in the degradative autophagy pathway. α-Mannosidase (Ams1) is another resident hydrolase that enters the vacuole independent of the secretory pathway; however, its mechanism of vacuolar delivery has not been established. We show vacuolar localization of Ams1 is blocked in mutants that are defective in the Cvt and autophagy pathways. We have found that Ams1 forms an oligomer in the cytoplasm. The oligomeric form of Ams1 is also detected in subvacuolar vesicles in strains that are blocked in vesicle breakdown, indicating that it retains its oligomeric form during the import process. These results identify Ams1 as a second biosynthetic cargo protein of the Cvt and autophagy pathways. endoplasmic reticulum vacuolar α-mannosidase autophagy aminopeptidase I carboxypeptidase Y cytoplasm to vacuole targeting N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide hemagglutinin epitope precursor aminopeptidase I synthetic minimal medium lacking nitrogen synthetic minimal medium containing 3% glycerol synthetic minimal medium containing nitrogen yellow fluorescent protein yeast nitrogen base n-octyl-β-d-glucopyranoside 2-(N-morpholino)ethanesulfonic acid 3-(N-morpholino)propanesulfonic acid kilobase pair(s) polyacrylamide gel electrophoresis Tris salts buffer One of the hallmarks of eukaryotic cells is the presence of a variety of membrane enclosed organelles. These compartments are critical to the proper physiology of the cell. In order to ensure correct function, cells must maintain faithful sorting of resident proteins to each organelle. A substantial amount of information is known about the delivery of proteins to compartments of the endomembrane system including the endoplasmic reticulum (ER),1 Golgi complex, and vacuole/lysosome. In brief, proteins can enter the ER post-translationally through proteinaceous channels and transit to other compartments through transient carrier vesicles. However, other compartments are not able to receive proteins through this route. For example, peroxisomes, mitochondria, and chloroplasts maintain a separate mechanism(s) for translocating proteins across their membranes (1Keegstra K. Froehlich J.E. Curr. Opin. Plant Biol. 1999; 2: 471-476Crossref PubMed Scopus (62) Google Scholar, 2Rassow J. Pfanner N. Traffic. 2000; 1: 457-464Crossref PubMed Scopus (34) Google Scholar, 3Subramani S. Koller A. Snyder W.B. Annu. Rev. Biochem. 2000; 69: 399-418Crossref PubMed Scopus (200) Google Scholar). One challenge facing eukaryotic cells is the import of large assembled protein complexes into organelles. Although several organelles have the capacity to translocate folded proteins (4Teter S.A. Klionsky D.J. Trends Cell Biol. 1999; 9: 428-431Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), in most cases complexes assemble subsequent to import of the individual protein monomers. In contrast, the peroxisome has the capacity for importing oligomeric proteins directly from the cytosol (5McNew J.A. Goodman J.M. J. Cell Biol. 1994; 127: 1245-1257Crossref PubMed Scopus (278) Google Scholar). Several components in peroxisome protein import are currently being characterized, but the actual mechanics of import are not defined (3Subramani S. Koller A. Snyder W.B. Annu. Rev. Biochem. 2000; 69: 399-418Crossref PubMed Scopus (200) Google Scholar). Both a transient pore/shuttle or a vesicular invagination of the peroxisome membrane could act in conjunction with the known receptor proteins. Although most vacuolar proteins enter the vacuole through a portion of the secretory pathway, the resident hydrolase aminopeptidase I (API) uses an alternative process (6Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Crossref PubMed Scopus (305) Google Scholar). Precursor API (prAPI) is synthesized in the cytosol on ribosomes that are not attached to the ER. Following synthesis, prAPI rapidly assembles into a dodecamer and subsequently into a higher order complex (7Kim J. Scott S.V. Oda M.N. Klionsky D.J. J. Cell Biol. 1997; 137: 609-618Crossref PubMed Scopus (116) Google Scholar). This complex of dodecamers becomes sequestered by membrane, resulting in the formation of a double-membrane cytosolic vesicle (8Scott S.V. Baba M. Ohsumi Y. Klionsky D.K. J. Cell Biol. 1997; 138: 37-44Crossref PubMed Scopus (141) Google Scholar, 9Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (277) Google Scholar). Upon completion, this vesicle fuses with the vacuole to release a single-membrane vesicle into the lumen. Following breakdown of the vesicle, prAPI is matured by removal of its propeptide. This import process is termed the cytoplasm to vacuole targeting (Cvt) pathway. The Cvt pathway has been shown to overlap with autophagy (10Harding T.M. Hefner-Gravink A. Thumm M. Klionsky D.J. J. Biol. Chem. 1996; 271: 17621-17624Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 11Scott S.V. Hefner-Gravink A. Morano K.A. Noda T. Ohsumi Y. Klionsky D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12304-12308Crossref PubMed Scopus (213) Google Scholar). Autophagy is a degradative process that employs similar changes in membrane topology to sequester cytosol for breakdown and recycling under starvation conditions. During starvation, the level of prAPI increases and the protein is imported to the vacuole via autophagosomes (9Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (277) Google Scholar). An intriguing question has been why yeast cells have utilized such a complex process as the Cvt pathway for the import of a resident vacuolar hydrolase. One possibility is that the dodecameric structure of prAPI is critical for its stability and/or function. The size of the dodecamer prevents translocation through the ER translocon, necessitating a vesicle-mediated import process. In addition, it has not been known whether other resident hydrolases utilize the Cvt pathway for vacuolar localization. α-Mannosidase (Ams1) is a resident vacuolar hydrolase that has been shown to enter the vacuole independent of the secretory pathway (12Yoshihisa T. Anraku Y. J. Biol. Chem. 1990; 265: 22418-22425Abstract Full Text PDF PubMed Google Scholar). As a resident vacuolar hydrolase, one role of Ams1 is to aid in recycling macromolecular components of the cell through hydrolysis of terminal, non-reducing α-d-mannose residues (EC3.2.1.24). In growing wild type cells, a relatively low basal level of Ams1 is synthesized. Under conditions of greater recycling need (i.e. nutrient deprivation), Ams1 levels are induced, and this coincides with induction of the nonspecific delivery mechanism of autophagy. We show in this report that Ams1 forms an oligomer in the cytoplasm. Under nutrient-rich conditions, oligomeric Ams1 is delivered to the vacuole by the Cvt pathway. Under starvation conditions, the up-regulated oligomeric protein is localized through autophagy. Mutants in either pathway are defective in Ams1 import. These results define Ams1 as a second cargo protein that utilizes both the Cvt and autophagic pathways for biosynthetic delivery to the vacuole. Prestained molecular weight markers were from Bio-Rad. Oligonucleotide primers were synthesized by Operon Technologies (Alameda, CA). The pAM1 plasmid (13Kuranda M.J. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2585-2589Crossref PubMed Scopus (77) Google Scholar) was a gift from Drs. Michael J. Kuranda and Phillips W. Robbins (Massachusetts Institute of Technology, Cambridge, MA). Yeast nitrogen base (YNB) was from Difco. YNB without copper ions was from BIO 101 (Vista, CA). Ficoll, DEAE-dextran, and the Vistra ECF Western blotting reagents were from Amersham Pharmacia Biotech. Restriction enzymes, ligase, and DNA polymerase were from New England Biolabs (Beverly, MA). Yeast lytic enzyme was from ICN Biomedicals (Aurora, OH). Complete EDTA-free protease inhibitors were from Roche Molecular Biochemicals.n-Octyl-β-d-glucopyranoside detergent (βOG) was from Calbiochem (La Jolla, CA).N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM 4–64) was from Molecular Probes (Eugene, OR). Polyclonal antiserum to the HA epitope was from Santa Cruz Biotechnology (Santa Cruz, CA). Other reagents were from Sigma unless noted. Strains used were SEY6210 MATα ura3–52 leu2–3,112 his3-Δ200 trp1-Δ901 lys2–801 suc2-Δ9 mel GAL (14Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (732) Google Scholar); JKY007, SEY6210agp9Δ (15Noda T. Kim J. Huang W.-P. Baba M. Tokunaga C. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; 148: 465-479Crossref PubMed Scopus (304) Google Scholar); AHY1468, SEY6210 apg1/cvt10–4; THY193, SEY6210 apg7/cvt2–1; AHY1293, SEY6210apg14/cvt12–1; THY313, SEY6210 aut7/cvt5–1; THY119, SEY6210 cvt3–1; AHY96, SEY6210 cvt9–1; THY32, SEY6210 cvt17–1 (10Harding T.M. Hefner-Gravink A. Thumm M. Klionsky D.J. J. Biol. Chem. 1996; 271: 17621-17624Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 16Harding T.M. Morano K.A. Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 591-602Crossref PubMed Scopus (397) Google Scholar). MHY10 (SEY6210pep4Δ::URA3) was generated as described for MHY8 (17Hutchins M.U. Veenhuis M. Klionsky D.J. J. Cell Sci. 1999; 112: 4079-4087Crossref PubMed Google Scholar). THY411 (SEY6210ams1Δ::LEU2) was generated by transforming yeast with PvuII-linearized pDelLAMS (described below), which replaces the first third of the chromosomalAMS1 gene with LEU2. The 3×HA-tagged Ams1 strain MHY11 was generated by the integration of a DNA fragment encoding the 3×HA epitope at the 3′ end of the chromosomal AMS1 locus in strain SEY6210 using the ME3 plasmid (provided by Dr. Neta Dean, State University of New York, Stony Brook, NY) as a template (primers: AMSPREHA (5′-AATTGAGACCTTTTGAGATTGCCTCATTCAGGTTGTATTTCTACCCATACGATGTTCCT-3′) and AMSpastHA (5′-TTTACTTATATGTATTTTGTTAAGACTATTTTTGGTTATCAGTCGACGGTATCGATAAG-3′)). Confirmation of the integratedAMS1::AMS1-HA was shown by induction of a ∼132-kDa fusion protein. Integration of the HA epitope at the chromosomal AMS1 locus had no detectable effect on Ams1 activity or its vacuolar localization (data not shown). Strains were typically grown on SMD (0.67% YNB, 2% glucose). Strains were also grown to a density of 0.5 A600 in SMD and were resuspended and grown in SGd (0.67% YNB, 3% glycerol, 0.1% glucose) for induction of Ams1 where indicated. Strains that were induced with 10 or 50 μm copper sulfate were first subcultured three times in SMD lacking copper ions. All media were brought to pH 5.5 with 50 mm each MES and MOPS and supplemented with required amino acids and vitamins. The copper-inducible AMS1 plasmids pCuAMS1(414) and pCuAMS1(424) (pMUH25 and pMUH26, respectively) were generated as follows. Oligonucleotide primers (AM1LOWSPE, 5′-CAAAAACTAGTAATTATGTCATC-3′; and AM1UP2, 5′-CGCTTGCTCGAGCTCCTCTATGTGGATAG-3′) were used to amplify a truncatedAMS1 sequence from pAM1 (13Kuranda M.J. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2585-2589Crossref PubMed Scopus (77) Google Scholar). The 2.9-kb product was ligated into pCR-Blunt (Invitrogen, Carlsbad, CA), resulting in a 6.4-kb construct with flanking SpeI sites (pMUH18). pMUH18 was digested with SpeI, and the resulting 2.9-kb fragment was ligated in the correct orientation at the SpeI site of plasmid pCu414 (provided by Dr. Dennis J. Thiele, University of Michigan, Ann Arbor, MI; Ref. 18Labbé S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), resulting in a 7.8-kb plasmid (pMUH21). To restore the full-length sequence of AMS1, theAMS1 gene was amplified from the pAM1 plasmid (primers: AM1LOWER, 5′-CCTAACTCGTTTAAGGGAGAC-3′; and AM1UPPER, 5′-CAGTGAGGGAGACAAACTCAG-3′). A 3.4-kb product was ligated into pCR-Blunt to generate a 6.9-kb plasmid (pMUH10). The 3.5-kbXhoI/SpeI fragment from pMUH10 was ligated into the same sites of pRS423 (19Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) generating pMUH12. The carboxyl terminus of AMS1 was restored with the 525-base pairSphI/XhoI fragment from pMUH12 ligated into pMUH21 resulting in the centromeric (CEN) plasmid pCuAMS1(414) (pMUH25). The multicopy (2μ) plasmid pCuAMS1(424) (pMUH26) was generated by ligating the 2.9-kb SpeI fragment from pMUH18 into the SpeI site of pCu424 (18Labbé S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), resulting in an 8.5-kb plasmid (pMUH22). The carboxyl terminus of AMS1 was restored as described for pMUH25. pCuAMS1YFP (pMUH29) was generated from pMUH21 and the 780-base pair fragment from pEYFP (CLONTECH, Palo Alto, CA), each digested with SphI/EcoRI. The resulting fusion protein contained the amino-terminal 929 amino acid residues from Ams1 followed by a 14-amino acid linker and YFP sequence. The ams1Δ::LEU2 deletion plasmid, pDelLAMS1, was generated from a 1-kb upstream portion and a 1.5-kb interior portion of the AMS1 gene isolated separately from pAM1 by a XhoI/HindIII digest and aBglII/EcoRI/HindIII digest, respectively. These fragments were ligated in the vector pRS306 (19Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) resulting in a unique HindIII site between the two segments of AMS1. This plasmid was digested with HindIII, and a HindIII fragment containing the LEU2 gene was cloned into this site to generate plasmid pDelLAMS1. The plasmid pCYI-50 (20Johnson L.M. Bankaitis V.A. Emr S.D. Cell. 1987; 48: 875-885Abstract Full Text PDF PubMed Scopus (179) Google Scholar) encodes the first 50 amino acids of carboxypeptidase Y (CPY) including the vacuolar targeting signal, fused to the periplasmic protein invertase, lacking its amino-terminal signal sequence in the vector pSEYC306. The plasmid pLJL2 is similar to pCYI-50 but carries the LEU2 gene instead ofURA3. Vacuoles were isolated on a Ficoll step gradient essentially as described previously (21Haas A. Methods Cell Sci. 1995; 17: 283-294Crossref Scopus (99) Google Scholar) with minor adaptations. Cells were grown in SMD to early log phase, and then ∼500 A600 units were harvested and spheroplasted with 6–10 mg of yeast lytic enzyme. Ninety percent or greater spheroplasting efficiency was determined by comparing theA600 of cells diluted 1:10 in water before and after addition of lytic enzyme. The plasma membrane was lysed using 400 μl of a 0.4 mg/ml solution of DEAE-dextran by being chilled on ice for 2 min, shaken at 30 °C for 2.5 min, and placed on ice again. 3 ml of lysed cell solution containing 2 mmphenylmethylsulfonyl fluoride were loaded at the bottom of an ultraclear SW41 tube (Beckman Coulter, Fullerton, CA) and overlaid with 8%, 4%, and 0% Ficoll solutions. Vacuoles were collected from the 0%/4% float interface with a Pasteur pipette after a 1.5-h spin at 30,000 × g. Enzyme assays for marker proteins were performed on material loaded onto the gradient and the recovered vacuole float fraction. Cell lysates and harvested vacuoles were assayed for marker enzymes invertase, α-glucosidase, and NADPH-cytochrome c reductase as described (20Johnson L.M. Bankaitis V.A. Emr S.D. Cell. 1987; 48: 875-885Abstract Full Text PDF PubMed Scopus (179) Google Scholar). Ams1 activity was determined based on established protocol (22Opheim D.J. Biochim. Biophys. Acta. 1978; 524: 121-130Crossref PubMed Scopus (69) Google Scholar). Samples were treated with Triton X-100 (2.5% final concentration), and then the volume was brought up to 400 μl with distilled H2O. 100 μl of 5× substrate mix (200 mm sodium acetate, pH 6.5, 2 mm p-nitrophenyl-α-d-mannopyranoside) was added to start the reaction and was incubated for 1 h at 37 °C. The reaction was stopped with 200 μl of 10% trichloroacetic acid, and any particulates were spun down in a microcentrifuge for 5 min. An equal volume of 1 m glycine, pH 10.4, was added to neutralize the reaction before the absorbance at 400 nm was read. Results from assays for each strain were tabulated from a minimum of four independent vacuole preparations. Antisera was generated to Ams1 using two synthetic peptides (Multiple Peptide Systems, San Diego, CA) corresponding to amino acid residues 79–96 and residues 991–1011. These peptides were conjugated to keyhole limpet hemocyanin and injected into a New Zealand White male rabbit using standard procedures. Immunoblots were prepared from 8% SDS-PAGE gels as described (16Harding T.M. Morano K.A. Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 591-602Crossref PubMed Scopus (397) Google Scholar) with 15% methanol in the transfer buffer. Ams1 antiserum was blocked by incubating with ams1Δ cell extract in TTBS (20 mm Tris, pH 7.6, 0.8% NaCl, 0.1% (w/v) Tween 20) for 2–3 h at 4 °C prior to use at a 1:12,500 dilution for 4 h at room temperature. Polyclonal antiserum to the HA epitope was used at a 1:1,000 dilution. API antiserum was described previously (6Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Crossref PubMed Scopus (305) Google Scholar). Quantitation by Vistra kit or use of secondary antibodies conjugated to horseradish peroxidase was as described (16Harding T.M. Morano K.A. Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 591-602Crossref PubMed Scopus (397) Google Scholar,17Hutchins M.U. Veenhuis M. Klionsky D.J. J. Cell Sci. 1999; 112: 4079-4087Crossref PubMed Google Scholar). Immunoblots were quantified on a STORM PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Strains harboring the pCuAMS1(414) plasmid encoding AMS1 under a copper regulable promoter were grown in SMD in the absence of copper. The presence of this plasmid facilitated the visualization of Ams1 under the indicated growth conditions. Cells were induced with 50 μmCuSO4 for 1 h. Twenty A600units were harvested in log phase and washed in Tris salts buffer (TSB; 50 mm Tris, pH 8.5, 50 mm KOAc, 100 mm KCl, 0.5 mm MgCl2). Cells were transferred to a microcentrifuge tube and resuspended in 250 μl of TSB containing 1 μg/ml pepstatin A and Complete EDTA-free protease inhibitors. Cells were lysed with glass beads for 1 min, and then βOG was added to 2% final concentration and mixed gently with a pipette tip. 200 μl of sample was loaded on a 1.8-ml prepoured 20–50% glycerol gradient made up in TSB and containing protease inhibitors. Oligomeric complexes were resolved at 55,000 rpm for 4 h in a Beckman TLS-55 rotor at 15 °C. Ten supernatant fractions of 200 μl were collected from the top of the gradient and precipitated with 10% trichloroacetic acid on ice. Fraction 10 may contain some amount of contaminating pellet material. Samples were washed twice in acetone and sonicated in MES/urea resuspension buffer (6Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Crossref PubMed Scopus (305) Google Scholar) before resolving by SDS-PAGE, followed by immunoblot and detection of Ams1 or API. A higher molecular mass cross-reactive protein detected by Ams1 antiserum (see Fig.1 B, lane 5) migrated to fractions 3–4 in wild type and cvt2 cells (Fig. 3, A andB). Quantification of these fractions was arbitrarily set to zero for clarity. Protein standards were run on an identical gradient and the peak fraction for each was determined by Coomassie Brilliant Blue staining of a SDS-PAGE gel. Bovine serum albumin (66 kDa), aldolase (158 kDa), and catalase (240 kDa), peaked in fractions 3, 4, and 5, respectively, whereas apoferritin (450 kDa), urease (545 kDa) and thyroglobulin (669 kDa) all peaked in fraction 6 of the gradient.Figure 3Ams1 transits to the vacuole as an oligomer. A, wild type (WT) cells harboring the CEN Ams1 plasmid pCuAMS1(414) were grown to early log phase and induced with 50 μm copper sulfate for 1 h. Strain MHY11 (AMS1::AMS1-HA) was grown to early log phase and harvested directly. 20 A600units of cells were lysed with glass beads, mixed with βOG detergent (2% final concentration), and loaded on a 20–50% glycerol gradient. Ten fractions of 200 μl were collected from the top and examined by immunoblot. Immunoblots were probed with anti-Ams1, anti-HA, or anti-API antisera and then quantitated using the Vistra detection reagents as described under "Experimental Procedures." Ams1 peaked as an oligomeric complex in fraction 6, whereas mAPI peaked at fraction 7 (7Kim J. Scott S.V. Oda M.N. Klionsky D.J. J. Cell Biol. 1997; 137: 609-618Crossref PubMed Scopus (116) Google Scholar). Protein standards run on an identical gradient included: bovine serum albumin (66 kDa, fraction 3), aldolase (158 kDa, fraction 4), catalase (240 kDa, fraction 5), apoferritin (450 kDa, fraction 6), urease (545 kDa, fraction 6), and thyroglobulin (669 kDa, fraction 6). Note that the diffuse band in fractions 3 and 4 of the gradient detected with peptide antiserum is a cross-reacting contaminant and was not included in the quantification. B and C, oligomerization of Ams1 occurs in the cytosol and is maintained during vacuolar delivery. Native protein extracts from the apg7 andcvt17 mutant strains harboring pCuAMS1(414) were analyzed by glycerol gradients as in A. Ams1 is an oligomer inapg7, a mutant that is defective in vacuolar delivery of Ams1, and in cvt17 that is defective in the breakdown of Cvt bodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For separation of the MHY11 vacuole fraction on a glycerol gradient, purified vacuoles were collected as described above and 10× TSB was added to 1× prior to a 5-min, 4 °C centrifugation step at 13,000 rpm. The pellet containing the concentrated vacuoles was resuspended in 250 μl of TSB plus inhibitors and detergent and mixed, and 200 μl was loaded onto the gradient and collected as above. Confocal microscopy (Leica IRM confocal microscope) images were taken as an average of 4–8 scans of a single focal plane. Cells harboring the pCuAMS1YFP plasmid (pMUH29) were grown to early log phase and induced with 10 μmcopper sulfate for 12 h. 1 ml of cells were then harvested and resuspended with 100 μl of fresh medium containing 4–8 μm FM 4–64 (23Vida T.A. Emr S.D. J. Cell Biol. 1995; 128: 779-792Crossref PubMed Scopus (1132) Google Scholar) for 30 min to label the vacuoles. FM 4–64 was chased to the vacuole for 1 h by the addition of 1 ml of fresh medium. Cells were then washed once in SD−N (containing 10 μm copper sulfate) and incubated in fresh SD−N for 12 h. Most of the characterized vacuolar hydrolases are proteolytically processed upon delivery to the vacuole. The resulting shift in molecular mass provides a convenient means for monitoring delivery to the organelle. We were interested in determining the mechanism of vacuolar delivery used by the resident hydrolase α-mannosidase (Ams1). However, previous analyses of Ams1 have been equivocal with regard to whether this protein is rapidly processed following delivery to the vacuole (12Yoshihisa T. Anraku Y. J. Biol. Chem. 1990; 265: 22418-22425Abstract Full Text PDF PubMed Google Scholar, 24Yoshihisa T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1988; 263: 5158-5163Abstract Full Text PDF PubMed Google Scholar). To examine processing of Ams1, we generated antiserum to synthetic peptides corresponding to the deduced amino acid sequence as described under "Experimental Procedures." We were unable to detect Ams1 synthesized from the chromosomal locus when cells were grown in minimal medium (SMD). This result was not surprising because Ams1 is made at very low levels in the presence of glucose unless cells are subjected to heat shock (12Yoshihisa T. Anraku Y. J. Biol. Chem. 1990; 265: 22418-22425Abstract Full Text PDF PubMed Google Scholar, 22Opheim D.J. Biochim. Biophys. Acta. 1978; 524: 121-130Crossref PubMed Scopus (69) Google Scholar, 24Yoshihisa T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1988; 263: 5158-5163Abstract Full Text PDF PubMed Google Scholar). However, when Ams1 was synthesized from a CEN or 2μ plasmid, we were able to detect a specific band in a dosage-dependent manner (Fig.1 A). Ams1 has a predicted molecular mass of 124 kDa. Previous studies indicated that the initial protein product was 107 kDa and that this was processed to 73 and 31 kDa forms that co-purified (24Yoshihisa T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1988; 263: 5158-5163Abstract Full Text PDF PubMed Google Scholar). However, we detected a band corresponding to Ams1 that migrated at 122 kDa (Fig.1 B) that is closer in agreement with the expected molecular mass and probably corresponds with the full-length protein. To increase the level of Ams1, we expressed the protein from a CEN plasmid under control of the CUP1 promoter. A 73-kDa cleavage product was not detected in wild type cells grown in SMD (Fig. 1 B,lane 1). After cells were grown to early log phase and shifted to glycerol medium (SGd), a 73-kDa protein was apparent that first appeared ∼12 h after the shift. This band increased in abundance from 12 to 24 h (Fig. 1 B,lanes 2 and 3). Appearance of the 73-kDa species was not seen in a pep4Δ strain (Fig.1 B, lane 4), indicating that it was due to a vacuole-dependent cleavage event in agreement with previous studies (12Yoshihisa T. Anraku Y. J. Biol. Chem. 1990; 265: 22418-22425Abstract Full Text PDF PubMed Google Scholar). However, the slow kinetics of cleavage and the observation that processing was incomplete, coupled with the fact that this processing event is not required for Ams1 enzymatic activity (12Yoshihisa T. Anraku Y. J. Biol. Chem. 1990; 265: 22418-22425Abstract Full Text PDF PubMed Google Scholar), suggest that the cleavage event may be a byproduct of vacuolar localization. Accordingly, processing of Ams1 is not a good indicator for a kinetic analysis of vacuolar delivery. Because Ams1 is not processed concomitant with its delivery to the vacuole, we could not rely on a mobility shift to follow localization. As an alternative approach, we analyzed its localization through subcellular fractionation. Yeast cells were grown to early log phase in minimal medium, and vacuoles were isolated on a Ficoll step gradient as described under "Experimental Procedures." The efficiency of vacuolar recovery was determined by assessing the invertase activity of a vacuolar targeted fusion protein consisting of a portion of the resident vacuolar protease CPY fused to the marker protein invertase (20Johnson L.M. Bankaitis V.A. Emr S.D. Cell. 1987; 48: 875-885Abstract Full Text PDF PubMed Scopus (179) Google Scholar). This chimeric construct utilizes the vacuolar targeting signal in CPY to divert the periplasmic enzyme invertase from the secretory pathway to the vacuole. The percentage of invertase activity in the purified vacuole fraction relative to the activity in a total spheroplast lysate provides a measure for the efficiency of organelle purification. Vacuole recovery of the CPY-invertase hybrid protein typically was 20% or higher as compared with the activity in the total fraction. In wild type cells, Ams1 activity in the purified vacuolar fraction was essentially equivalent to the vacuolar recovery based on invertase activity from the CPY-invertase marker (Fig.2 A). This result indicates that vacuole purification is an effective method for monitoring vacuolar delivery of Ams1. We next examined whether Ams1 was delivered to the vacuole in strains defective specifically in the Cvt pathway. The cvt3 andcvt9 mutants accumulate the precursor form of API but are relatively normal for autophagy (11Scott S.V. Hefner-Gravink A. Morano K.A. Noda T. Ohsumi Y. Klionsky D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12304-12308Crossref PubMed Scopus (213) Google Scholar, 25Kim J. Kamada Y. Stromhaug P.E. Guan J. Hefner-Gravink A. Baba M. Scott S.V. Ohsumi Y. Dunn Jr., W.A.