Ald6p Is a Preferred Target for Autophagy in Yeast, Saccharomyces cerevisiae
2004; Elsevier BV; Volume: 279; Issue: 16 Linguagem: Inglês
10.1074/jbc.m312706200
ISSN1083-351X
AutoresJun Onodera, Yoshinori Ohsumi,
Tópico(s)Biofuel production and bioconversion
ResumoMacroautophagy is the process of intracellular bulk protein degradation induced by nutrient starvation and is generally considered to be a nonselective degradation of cytosolic enzymes and organelles. However, it remains a possibility that some proteins may be preferentially degraded by autophagy. In this study, we have performed a systematic analysis on the substrate selectivity of autophagy in yeast, Saccharomyces cerevisiae, using two-dimensional PAGE. We performed a differential screen on wild-type and Δatg7/apg7 autophagy-deficient cells and found that cytosolic acetaldehyde dehydrogenase (Ald6p) decreased under nitrogen starvation. As assessed by immunoblot, Ald6p was reduced by greater than 82% after 24 h of nitrogen starvation. This reduction was dependent on Atg/Apg proteins and vacuolar proteases but was not dependent on the proteasome or the cytoplasm to vacuole targetting (Cvt) pathway. Using pulse-chase and subcellular fractionation, we have also demonstrated that Ald6p was preferentially transported to vacuoles via autophagosomes. Δatg7 Δald6 double mutant cells were able to maintain higher rates of viability than Δatg7 cells under nitrogen starvation, and Ald6p-overexpressing cells were not able to maintain high rates of viability. Furthermore, the Ald6pC306S mutant, which lacks enzymatic activity, had viability rates similar to Δald6 cells. Ald6p enzymatic activity may be disadvantageous for survival under nitrogen starvation; therefore, yeast cells may preferentially eliminate Ald6p via autophagy. Macroautophagy is the process of intracellular bulk protein degradation induced by nutrient starvation and is generally considered to be a nonselective degradation of cytosolic enzymes and organelles. However, it remains a possibility that some proteins may be preferentially degraded by autophagy. In this study, we have performed a systematic analysis on the substrate selectivity of autophagy in yeast, Saccharomyces cerevisiae, using two-dimensional PAGE. We performed a differential screen on wild-type and Δatg7/apg7 autophagy-deficient cells and found that cytosolic acetaldehyde dehydrogenase (Ald6p) decreased under nitrogen starvation. As assessed by immunoblot, Ald6p was reduced by greater than 82% after 24 h of nitrogen starvation. This reduction was dependent on Atg/Apg proteins and vacuolar proteases but was not dependent on the proteasome or the cytoplasm to vacuole targetting (Cvt) pathway. Using pulse-chase and subcellular fractionation, we have also demonstrated that Ald6p was preferentially transported to vacuoles via autophagosomes. Δatg7 Δald6 double mutant cells were able to maintain higher rates of viability than Δatg7 cells under nitrogen starvation, and Ald6p-overexpressing cells were not able to maintain high rates of viability. Furthermore, the Ald6pC306S mutant, which lacks enzymatic activity, had viability rates similar to Δald6 cells. Ald6p enzymatic activity may be disadvantageous for survival under nitrogen starvation; therefore, yeast cells may preferentially eliminate Ald6p via autophagy. Cellular activities require the maintenance of a balance between the synthesis and degradation of proteins. Macroautophagy (hereafter referred to as autophagy) is an intracellular bulk degradation system that is well conserved in eukaryotes; autophagy transports cytoplasmic components to the lysosome/vacuole for degradation (1Ashford T.P. Porter K.R. J. Cell Biol. 1962; 12: 198-202Crossref PubMed Scopus (445) Google Scholar). This degradation is a cellular response to starvation and also plays a role in the recycling of cytoplasmic components, which may be important for cellular remodeling, development, and differentiation (2Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1390) Google Scholar, 3Otto G.P. Wu M.Y. Kazgan N. Anderson O.R. Kessin R.H. J. Biol. Chem. 2003; 278: 17636-17645Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 4Melendez A. Talloczy Z. Seaman M. Eskelinen E.L. Hall D.H. Levine B. Science. 2003; 301: 1387-1391Crossref PubMed Scopus (1017) Google Scholar). A total of 16 genes that are essential for autophagy and that are named APG and AUT (current nomenclature is ATG) (5Klionsky D.J. Cregg J.M. Dunn W.A. Emr S.D. Sakai Y. Sandoval I.V. Sibirny A. Subramani S. Thumm M. Veenhuis M. Ohsumi Y. Dev. Cell. 2003; 5: 539-545Abstract Full Text Full Text PDF PubMed Scopus (1005) Google Scholar) have been identified by genetic screens in yeast, Saccharomyces cerevisiae. Much progress has been made in the functional analysis of these genes (6Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (389) Google Scholar, 7Ohsumi Y. Nat. Rev. Mol. Cell. Biol. 2001; 2: 211-216Crossref PubMed Scopus (1035) Google Scholar, 8Barth H. Meiling-Wesse K. Epple U.D. Thumm M. FEBS Lett. 2001; 508: 23-28Crossref PubMed Scopus (91) Google Scholar). In eukaryotic cells, there is another major degradation system, the ubiquitin proteasome pathway, which mediates the selective ubiquitination and subsequent degradation of proteins by the proteasome. This pathway serves mainly to degrade short-lived proteins, such as transcription factors, cell cycle regulators, and defective proteins. However, more than 99% of cellular proteins are long-lived, and autophagic degradation contributes to the turnover of these proteins. In contrast to selective degradation by the ubiquitin proteasome pathway, autophagy is generally thought to be nonselective. Autophagy is initiated by the sequestration of cytoplasmic components in a double-membrane structure termed the autophagosome. Immunoelectron microscopy has shown that ribosomes and typical cytosolic marker enzymes, such as alcohol dehydrogenase (ADH) 1The abbreviations used are: ADH, alcohol dehydrogenase; API, aminopeptidase I; GFP, green fluorescent protein; MALDI-TOF, matrix-associated laser deionization time-of-flight; PGK, phosphoglycerate kinase.2 T. Noda and Y. Ohsumi, unpublished results. 1The abbreviations used are: ADH, alcohol dehydrogenase; API, aminopeptidase I; GFP, green fluorescent protein; MALDI-TOF, matrix-associated laser deionization time-of-flight; PGK, phosphoglycerate kinase.2 T. Noda and Y. Ohsumi, unpublished results. and phosphoglycerate kinase (PGK), are present in the autophagosome and autophagic bodies at the same densities as in the cytosol (9Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (402) Google Scholar). The measurement of the enzymatic activities of these proteins also supports this conclusion (10Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (949) Google Scholar).2 If degradation of long-lived proteins is exclusively mediated by autophagy, all proteins might be expected to have similar lifetimes. However, long-lived proteins have a variety of lifetimes; therefore, the autophagic pathway might have some selectivity. It is known that fructose-1,6-bisphosphatase (via the vacuolar import and degradation, the Vid pathway) (6Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (389) Google Scholar, 11Hoffman M. Chiang H.L. Genetics. 1996; 143: 1555-1566Crossref PubMed Google Scholar) and the peroxisome (via pexophagy) (12Mukaiyama H. Oku M. Baba M. Samizo T. Hammond A.T. Glick B.S. Kato N. Sakai Y. Genes Cells. 2002; 7: 75-90Crossref PubMed Scopus (101) Google Scholar) are selectively transported from the cytoplasm to the vacuole and degraded. To investigate the possibility of selective autophagic degradation, we attempted to compare the amounts of each intracellular protein under growth and starvation conditions in yeast, S. cerevisiae. We performed a systematic analysis using two-dimensional PAGE and MALDI-TOF mass spectrometry to detect the autophagy-dependent degradation of intracellular proteins. During these analyses, we observed an interesting behavior of the Mg2+- and NADPH-dependent cytosolic acetaldehyde dehydrogenase (Ald6p), which catalyzes the conversion of acetaldehyde to acetate in the cytosol (acetaldehyde + NADP+ → acetate + NADPH) (13Meaden P.G. Dickinson F.M. Mifsud A. Tessier W. Westwater J. Bussey H. Midgley M. Yeast. 1997; 13: 1319-1327Crossref PubMed Scopus (74) Google Scholar). The S. cerevisiae genome encodes five or more different members of the aldehyde dehydrogenase family. Ald4p is the major K+- and NAD+-dependent mitochondrial acetaldehyde dehydrogenase (14Tessier W.D. Meaden P.G. Dickinson F.M. Midgley M. FEMS Microbiol. Lett. 1998; 164: 29-34Crossref PubMed Google Scholar), and Ald5p is a minor K+-dependent mitochondrial acetaldehyde dehydrogenase, which is induced when cells are grown in ethanol-containing medium (15Kurita O. Nishida Y. FEMS Microbiol. Lett. 1999; 181: 281-287Crossref PubMed Google Scholar). Ald2p and Ald3p are closely related cytosolic enzymes that are required for in vivo pantothenic acid biosynthesis via conversion of 3-aminopropanol to β-alanine (16White W.H. Skatrud P.L. Xue Z. Toyn J.H. Genetics. 2003; 163: 69-77PubMed Google Scholar). Ald4p and Ald6p function in the conversion of acetaldehyde to acetate, which is a key intermediate during fermentation of sugars and growth on ethanol and are consequently important for acetyl-CoA production. In contrast, Ald2p, Ald3p, and Ald5p may not contribute to the oxidation of acetaldehyde in vivo. Therefore, Ald6p is the only cytosolic acetaldehyde dehydrogenase in the yeast cell. We demonstrate here that Ald6p is degraded preferentially by autophagy and that reduction of Ald6p may improve viability rates under nitrogen starvation. Yeast Strains and Media—The S. cerevisiae strains used in this study are listed in Table I. Standard techniques were used for yeast manipulation (17Bruke D. Dawson D. Stearn T. Method in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000Google Scholar). Yeast cells were grown in YPD medium (1% yeast extract, 2% polypeptone, and 2% glucose) or SD + CA medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% casamino acid, 0.5% ammonium sulfate, and 2% glucose) supplemented with 0.002% adenine sulfate, 0.002% uracil, and 0.002% tryptophan if necessary. For nitrogen starvation, SD(-N) medium (0.17% yeast nitrogen base without ammonium sulfate and amino acids and with 2% glucose) was used.Table IYeast strains used in this studyStrainGenotypeSourceSEY6210MATα his3Δ200 leu2-3,112 lys2-801 trp1Δ901 ura3-52 suc2Δ9Ref. 40Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (732) Google ScholarKVY118SEY6210; Δatg7::HIS3Ref. 41Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-276Crossref PubMed Scopus (731) Google ScholarJOY676SEY6210; Δatg7::HIS3 Δald6::kanMX4This studyJOY674SEY6210; Δatg7::HIS3 Δald4::kanMX4This studyJOY617SEY6210; Δatg17::HIS3This studyTVY1SEY6210; Δpep4::LEU2Ref. 42Gerhardt B. Kordas T.J. Thompson C.M. Patel P. Vida T. J. Biol. Chem. 1998; 273: 15818-15829Abstract Full Text Full Text PDF PubMed Scopus (50) Google ScholarJOY6005SEY6210; Δpep4::LEU2 Δald6::ALD6-GFPThis studyJOY6006SEY6210; Δpep4::LEU2 Δatg7::HIS3 Δald6::ALD6-GFPThis studyJOY66SEY6210; Δald6::kanMX4This studyJOY64SEY6210; Δald4::kanMX4This studyJOY69SEY6210; Δatg11::URA3This studyJOY622SEY6210; Δvid22::kanMX4This studyKVY4SEY6210; Δypt7::LEU2Ref. 43Kihara A. Noda T. Ishihara N. Ohsumi Y. J. Cell Biol. 2001; 152: 519-530Crossref PubMed Scopus (800) Google ScholarYAK1SEY6210; Δypt7::HIS3 Δatg1::LEU2This studyWCG4aMATaleu2-3,112 ura3 his3-11,15Ref. 28Heinemeyer W. Gruhler A. Mohrle V. Mahe Y. Wolf D.H. J. Biol. Chem. 1993; 268: 5115-5120Abstract Full Text PDF PubMed Google ScholarWCG4-11aWCG4a; pre1-1Ref. 28Heinemeyer W. Gruhler A. Mohrle V. Mahe Y. Wolf D.H. J. Biol. Chem. 1993; 268: 5115-5120Abstract Full Text PDF PubMed Google Scholar Open table in a new tab Plasmid Construction—To create the glutathione S-transferase-Ald6p fusion construct (pJO1), the open reading frame of ALD6 (YPL061w) lacking the initiation codon (1.5 kb) was amplified by genomic PCR using the following primers: 5′-CGCGGATCCACTAAGCTACACTTTGACACTGC-3′ and 5′-CCGCTCGAGCAACTTAATTCTGACAGCTTTTACTTC-3′. This strategy incorporated novel BamHI and XhoI sites into the resulting DNA fragment, which was then cloned into the BamHI and XhoI sites of pGEX-4T-1 (Amersham Biosciences) to yield pJO1. To create the Ald6p-GFP genome integration vector (pJO-402), novel XbaI sites were added to the terminator sequence (0.5 kb) of ALD6 by genomic PCR amplification using the following primers: 5′-GCTCTAGATGTACCAACCTGCATTTCTTTC-3′ and 5′-GCTCTAGACGAAGAAGGATGTTATTATATG-3′. Novel XhoI and BamHI sites were added to the ALD6 promoter region (0.3 kb) and the ALD6 open reading frame lacking the stop codon (1.5 kb) by genomic PCR amplification using the following primers: 5′-CGCTCGAGCACCGACCATGTGGGCAAATTC-3′ and 5′-CGCGGATCCCAACTTAATTCTGACAGCTTTTAC-3′. BamHI sites were added to the open reading frame of modified GFP (S65T) lacking the initiation codon by PCR amplification using the following primers: 5′-CGCGGATCCGGTAAAGGAGAAGAACTTTTCACTGG-3′ and 5′-CGGGATCCTTACTTGTATAGTTCATCCATG-3′. The resulting DNA fragments were cloned into the pRS306 integration vector (18Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to yield pJO402. To create the Ald6p overexpression construct (pJO203), XhoI and BamHI sites were added to a sequence containing the ALD6 open reading frame (1.5 kb), the promoter region (0.3 kb), and the terminator sequence (0.5 kb) by genomic PCR amplification using the following primers: 5′-CCGCTCGAGCACCGACCATGTGGGCAAATTC-3′ and 5′-CGCGGATCCCGAAGAAGGATGTTATTATATGATCTC-3′. The resulting DNA fragment was cloned into the BamHI and XhoI sites of the pRS426 multicopy plasmid (18Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to yield pJO203. A QuikChange™ site-directed mutagenesis kit (Stratagene) was used to create the Ald6pC306S mutant overexpression construct (pJO213). To generate pJO213, the pJO203 plasmid was amplified by PCR with the following primers: 5′-AGAACGCTGGTCAAATTTCTTCCTCTGGTT-3′ and 5′-AACCAGAGGAAGAAATTTGACCAGCGTTCT-3′. The site of mutagenesis in pJO213 was confirmed by automated DNA sequencing. Two-dimensional PAGE and Peptide Mass Fingerprinting—The lysates were prepared by breaking yeast cells with glass beads in lysis buffer containing 50 mm Tris-HCl (pH 8.0), 1 mm EDTA, 1 mm phenylmethanesulfonyl fluoride, and protease inhibitor mixture (Roche Applied Science). The lysates were centrifuged at 100,000 × g for 1 h, and the supernatant was desalted with NAP-10™ (Amersham Biosciences). The protein concentrations were determined using a BCA assay kit (Pierce), and 300 μg of each lysate was applied to the gel. Isoelectric focusing was performed with IPGphor™ (Amersham Biosciences) and a 13 cm Immobiline™ DryStrip pH 4–7 (Amersham Biosciences) as described (19Görg A. Obermaier C. Boguth G. Harder A. Scheibe B. Wildgruber R. Weiss W. Electrophoresis. 2000; 21: 1037-1053Crossref PubMed Scopus (1634) Google Scholar). The gel strip was subjected to SDS-PAGE (12.5% acrylamide), and the gel was stained with Coomassie Brilliant Blue R-250. The protein spots were picked, washed with 100 mm ammonium bicarbonate, dehydrated with acetonitrile, and dried in an evaporator. The spots were digested in the gel with 0.5 mg/ml of trypsin (Promega) in 100 mm ammonium bicarbonate for 12 h at 30 °C. The digested peptides were extracted from the gel with 10% formic acid and 50% acetonitrile and desalted with the ZipTip™ C-18 (Millipore). The samples were mixed with α-cyano-4-hydroxy-cinnamic acid (Fluka) in a 2:1 ratio and were analyzed by MALDI-TOF mass spectrometry, REFLEX III (Bruker). The proteins were identified by searching the ProFound data base (www.129.85.19.192/). Antibodies—Ald6p-specific antibodies were prepared as follows. The pJO1 plasmid was transformed into Escherichia coli (DH5α), and transformants were grown in LB medium containing 50 μg/ml ampicillin to an A600 of 0.6. Recombinant protein expression was induced with 1 mm isopropyl-β-d-thiogalactopyranoside for an additional 6 h at 37 °C. The recombinant protein was separated by SDS-PAGE and simultaneously stained with gel code (Pierce). The protein band was excised from the gel and eluted with an electric current. The eluted protein-dye complex was used to immunize rabbits. Anti-Ald4p/6p antibodies were purchased from Rockland. Anti-ADH antibodies have been described previously (9Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (402) Google Scholar). Anti-PGK antibody was purchased from Molecular Probes. Anti-aminopeptidase I (API) antibodies were our laboratory stock. Immunoblotting of Total Cell Lysates—Whole cell lysates were prepared by disrupting cells with glass beads in lysis buffer. SDS-PAGE and immunoblotting were performed as described (20Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (712) Google Scholar). Pulse-Chase Experiments—The cells were cultured in YPD medium to an A600 of 1.0 at 30 °C and were then washed twice and suspended in SD(-N) medium. The cells were pulse-labeled for 30 min by adding 1 MBq of [35S]methionine/1 A600 unit and chased by adding 0.004% methionine and 0.003% cysteine at 30 °C. Immunoprecipitation was performed as described (21Ishihara N. Hamasaki M. Yokota S. Suzuki K. Kamada Y. Kihara A. Yoshimori T. Noda T. Ohsumi Y. Mol. Biol. Cell. 2001; 12: 3690-3702Crossref PubMed Scopus (292) Google Scholar). Light Microscopy—Fluorescence microscopy was performed using a Delta Vision microscope (Applied Precision) as described (22Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (794) Google Scholar). Subcellular Fractionation and Proteinase K Protection Assay—Yeast cells were converted to spheroplasts by treatment with 25 unit/ml of Zymolyase-100T (Seikagaku Corporation). The spheroplast lysates were separated into cell fractions as described previously (21Ishihara N. Hamasaki M. Yokota S. Suzuki K. Kamada Y. Kihara A. Yoshimori T. Noda T. Ohsumi Y. Mol. Biol. Cell. 2001; 12: 3690-3702Crossref PubMed Scopus (292) Google Scholar). To examine proteinase K sensitivity, each fraction without protease inhibitors was treated with 2 mg/ml proteinase K on ice for 30 min with or without 1% Triton X-100. The samples were precipitated with 10% trichloroacetic acid, washed once with cold acetone, resuspended in SDS sample buffer, and analyzed by SDS-PAGE and immunoblotting. Determination of Cell Viability—The cell viability was measured by phloxine B (final concentration 2 μg/ml) stain, and fluorescence microscopy was measured with a blue filter. Brightly fluorescent cells were counted as dead cells (2Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1390) Google Scholar). Screen for Proteins Reduced under Nitrogen Starvation—We investigated the expression profiles of soluble proteins before and after nitrogen starvation using two-dimensional PAGE. Using this method, we expected to be able to identify cellular proteins whose levels decreased during nitrogen starvation. The yeast cells were grown at 30 °C in YPD medium, were harvested at mid logarithmic phase (A600 = 1.0), and were washed twice with starvation medium. The cells were then transferred to SD(-N) medium and incubated for 24 h. We chose a long stress period of 24 h to observe obvious differences in protein expression; importantly, most of the cells were still viable at this time point (2Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1390) Google Scholar). Major protein spots on the gel were identified by the peptide mass fingerprinting method using MALDI-TOF mass spectrometry. In both wild-type (SEY6210) and autophagy-defective Δatg7/apg7 (KVY118) yeast cells, most proteins showed little change after starvation (Fig. 1A, lanes 1–4). However, several proteins showed increased levels after starvation, including stress-induced protein, heat shock protein, and quenching enzyme of reactive oxygen species (data not shown). Cytosolic acetaldehyde dehydrogenase (Ald6p) exhibited the most apparent decrease during starvation in wild-type cells (Fig. 1A, lane 5). Therefore, we focused on this protein for further analysis. Proteins Required for the Reduction of Ald6p under Nitrogen Starvation—Using immunoblot analyses, we attempted to determine which proteins are required for the reduction of Ald6p. In wild-type cells (SEY6210), the amount of Ald6p decreased in a nearly linear manner and was ultimately reduced to 18% of the original level after 24 h of starvation (Fig. 1B). In contrast, Ald6p levels decreased only slightly in Δatg7 mutant cells (KVY118). We next investigated whether the amount of Ald6p was reduced in various yeast strains that are defective in various steps of autophagy. Δatg7 (KVY118), Δatg17/apg17 (JOY617), and all Δatg/apg mutant cells tested showed a similar defect in the loss of Ald6p (parts shown in Fig. 1C, lanes 1–6). The decrease of Ald6p also required Ypt7p, a protein that is essential for the fusion of autophagosomes to vacuoles (20Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (712) Google Scholar), and Pep4p, vacuolar proteinase A (Fig. 1C, lanes 1, 2, and 7–10). The selective transport of vacuolar enzymes (via the Cvt pathway), such as API and α-mannosidase, is known to utilize all of the Apg/Atg proteins except Atg17p (6Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (389) Google Scholar, 23Khalfan W.A. Klionsky D.J. Curr. Opin. Cell Biol. 2002; 14: 468-475Crossref PubMed Scopus (58) Google Scholar). Atg11p/Cvt9p and Atg19p/Cvt19p function only in the Cvt vesicle formation and do not play a role in autophagosome formation (24Kim J. Kamada Y. Stromhaug P.E. Guan J. Hefner-Gravink A. Baba M. Scott S.V. Ohsumi Y. Dunn Jr., W.A. Klionsky D.J. J. Cell Biol. 2001; 153: 381-396Crossref PubMed Scopus (216) Google Scholar, 25Scott S.V. Guan J. Hutchins M.U. Kim J. Klionsky D.J. Mol. Cell. 2001; 7: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). In Δatg11 (JOY69) and Δatg19 mutant cells, Ald6p was reduced in a manner similar to that of wild-type cells under nitrogen starvation (Fig. 1C, lanes 1, 2, 11, and 12; data for Δatg19 not shown). As expected, another system of vacuolar transport, the Vid pathway (6Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (389) Google Scholar, 11Hoffman M. Chiang H.L. Genetics. 1996; 143: 1555-1566Crossref PubMed Google Scholar, 26Brown C.R. McCann J.A. Hung G.G. Elco C.P. Chiang H.L. J. Cell Sci. 2002; 115: 655-666Crossref PubMed Google Scholar), was not involved in this phenomenon (Fig. 1C, lanes 1, 2, 13, and 14). One mutant allele of the proteasome subunit PRE1 is pre1-1, which is frequently used for the following reasons: the pre1-1 mutation causes a defect in the degradation of short-lived proteins, ubiquitinated proteins (27Heinemeyer W. Kleinschmidt J.A. Saidowsky J. Escher C. Wolf D.H. EMBO J. 1991; 10: 555-562Crossref PubMed Scopus (357) Google Scholar, 28Heinemeyer W. Gruhler A. Mohrle V. Mahe Y. Wolf D.H. J. Biol. Chem. 1993; 268: 5115-5120Abstract Full Text PDF PubMed Google Scholar) and N-end rule substrates (29Richter-Ruoff B. Heinemeyer W. Wolf D.H. FEBS Lett. 1992; 302: 192-196Crossref PubMed Scopus (66) Google Scholar, 30Seufert W. Jentsch S. EMBO J. 1992; 11: 3077-3080Crossref PubMed Scopus (121) Google Scholar) at 30 °C. In pre1-1 mutant cells (WCG4–11a), Ald6p was decreased similarly to wild-type cells (WCG4a) under nitrogen starvation, indicating that Ald6p is not a substrate for proteasome-mediated degradation (Fig. 1D). Taken together, these mutant studies indicate that the reduction of Ald6p requires all of the Atg/Apg proteins and the processes of vacuolar proteolysis. However, Atg/Cvt proteins, Vid proteins, and proteasomal degradation are not involved in this phenomenon. Reduced Ald6p Levels Implied a Rapid Degradation under Nitrogen Starvation—We hypothesized that the decrease in Ald6p levels was the result of rapid degradation during nitrogen starvation. To examine this possibility, the kinetics of Ald6p degradation was measured by pulse-chase experiments. Wild-type (SEY6210) and Δatg7 (KVY118) cells were pulse-labeled for 30 min with [35S]methionine and chased with cold methionine and cysteine for 0, 3, 6, and 9 h. In wild-type cells, the Ald6p was rapidly degraded and was barely detectable after 6 h of chase (Fig. 2). In contrast, the degradation rate of Ald6p was clearly slower in Δatg7 mutant cells. In addition, ADH, a known nonselective marker of autophagy (9Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (402) Google Scholar), did not show rapid degradation like Ald6p (Fig. 2). The reduction of Ald6p levels implied a rapid degradation dependent on Atg7p during nitrogen starvation. These results suggest that Ald6p is transported to the vacuole and degraded much more rapidly than typical cytosolic proteins. Visualization of Ald6p under Nitrogen Starvation—The process of Ald6p vacuolar transport was visualized by expressing physiological levels of an Ald6p-GFP fusion protein from the authentic ALD6 promoter. Upon starvation, the vacuoles gradually became fluorescent. In addition, in Δpep4 cells (JOY6005), many bright dots, which were presumably autophagic bodies, were observed moving around in the vacuole (Fig. 3). In Δpep4 Δatg7 double mutant cells (JOY6006), no fluorescence was observed in the vacuoles, but rather, the cytosol was evenly stained (Fig. 3). Because Ald6p-GFP was transported to the vacuole in autophagic bodies during nitrogen starvation, we hypothesized that transport of Ald6p from the cytosol to the vacuole occurred via the autophagosome. Ald6p Was Preferentially Transported to the Vacuole via the Autophagosome—We previously reported that Δypt7 cells accumulate autophagosomes in the cytosol under nitrogen starvation (20Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (712) Google Scholar). Using proform of API as a selective cargo marker of autophagosomes, Ishihara et al. (21Ishihara N. Hamasaki M. Yokota S. Suzuki K. Kamada Y. Kihara A. Yoshimori T. Noda T. Ohsumi Y. Mol. Biol. Cell. 2001; 12: 3690-3702Crossref PubMed Scopus (292) Google Scholar) showed the low speed pellet (P13) fraction enriches the autophagosomes. So next we studied the behavior of Ald6p in Δypt7 cells (KVY4). Under growing conditions proform of API was exclusively resided in the high speed supernatant (S100), but under nitrogen starvation conditions a significant portion was recovered in the P13 fraction as reported (Ref. 21Ishihara N. Hamasaki M. Yokota S. Suzuki K. Kamada Y. Kihara A. Yoshimori T. Noda T. Ohsumi Y. Mol. Biol. Cell. 2001; 12: 3690-3702Crossref PubMed Scopus (292) Google Scholar and Fig. 4A). Similarly Ald6p was recovered in the P13 fraction only under nitrogen starvation condition (Fig. 4A). This fraction completely diminished in Δypt7 Δatg1/apg1 mutant (YAK1; Fig. 4B, lanes 5–8), indicating that a certain amount of Ald6p is in the autophagosomes. As shown in Fig. 4D, Ald6p and proform of API in P13 fraction were resistant to proteinase K treatment but were digested in the presence of 1% Triton X-100. This also supported the possibility that Ald6p is sequestered into autophagosomes. We also quantified the amount of Ald6p in the P13 fraction. Proform of API forms one or a few large complex named the Cvt complex in the cytosol and is taken up by an autophagosome at once (31Suzuki K. Kamada Y. Ohsumi Y. Dev. Cell. 2002; 3: 815-824Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). PGK is shown to be distributed evenly in the autophagosome, autophagic bodies, and cytosol (9Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (402) Google Scholar). As shown in Fig. 4C, Ald6p translocated to the P13 fraction much more efficiently than PGK (recovery in P13 fraction; Ald6p = 38.2 ± 2.1% n = 5; PGK = 14.9 ± 1.5% n = 5; Fig. 4C) but less than proform of API (67.2 ± 5.9% n = 5). Taken together, we concluded that Ald6p is preferentially sequestered into autophagosome, possibly in a manner different from the substrates for the Cvt pathway. Ald6p Enzymatic Activity May Be Disadvantageous during Nitrogen Starvation—Next, we examined the physiological relevance of the preferential degradation of Ald6p during nitrogen starvation. Autophagy-defective mutants cannot maintain viability under long span nitrogen starvation (2Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1390) Google Scholar). Δatg7 Δald6 mutant (JOY676) cells also started to die after 2 days of nitrogen starvation, but its viability decreased more slowly than that of Δatg7 mutant cells (KVY118; Fig. 5A). The viability of Atg+ Δald6 cells (JOY66) also improved slightly from that of wild-type cells (Atg+ALD6; SEY6210) under nitrogen starvation. However, disruption of mitochondrial acetaldehyde dehydrogenase (Ald4p), Atg+ald4 (JOY64), and Δatg7 Δald4 (JOY674) cells had no effect on the viabilities of wild-type (SEY6210) and Δatg7 (KVY118) cells, respectively (Fig. 5B). Furthermore, wild-type cells (Atg+) expressing Ald6p via multicopy plasmid showed a defect in the maintenance of viability during nitrogen starvation (Fig. 5C). These results indicate that abundant Ald6p causes the decrease of viability, and the absence of Ald6p improves viability under nitrogen starvation. We next asked whether Ald6p enzymatic activity or the protein molecule itself is harmful to the cell under nitrogen starvation. To address it we constructed an inactive Ald6p mutant. Farres et al. (32Farres J. Wang T.T. Cunningham S.J. Weiner H. Biochemistry. 1995; 34: 2592-2598Crossref PubMed Scopus (142) Google Scholar) isolated recombinant ALDH2C302S from rat liver mitochondrial class-2 aldehyde dehydrogenase (ALDH2). Rat ALDH2-Cys302 is an active site residue whose thiol group binds to the aldehyde group of the substrate. Ald6p-Cys306, which corresponds to Rat ALDH2-Cys302, was changed to a serine residue by site-directed mutagenesis. It was confirmed that the mutant form of Ald6p lost completely NADP+- and Mg2+-dependent acetaldehyde dehydrogenase activity (data not shown). Overexpression of Ald6pC306S in Atg+ and Δatg7 cells had no effect on viabilities of Atg+ Δald6 and Δatg7 Δald6, respectively (Fig. 5D). These results indicate that the acetaldehyde dehydrogenase activity of cytosolic Ald6p may have a disadvantageous effect on the survival of yeast cells during nitrogen starvation. We surveyed the changes of soluble proteins before and after nitrogen starvation using two-dimensional PAGE. One protein, Ald6p, showed a clear reduction, which was dependent on Atg/Apg proteins, under nitrogen starvation for 24 h (Fig. 1). Previous morphological studies have indicated that autophagy degrades about 2% of the cytosol/h in yeast (9Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (402) Google Scholar, 10Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (949) Google Scholar). Scott et al. (33Scott 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) showed by [35S]methionine pulse-chase experiments that the rate of vacuolar delivery of cytosolic Pho8Δ60p by autophagy was 4%/h during the initial6hof nitrogen starvation. Autophagy proceeds linearly during the first 6 h of starvation and then gradually slows (33Scott 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). We know that both diploid and haploid cells induce autophagy in sporulation medium, 2% potassium acetate (10Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (949) Google Scholar). In a previous report, Betz and Weiser (34Betz H. Weiser U. Eur. J. Biochem. 1976; 70: 385-395Crossref PubMed Scopus (26) Google Scholar) showed that protein degradation in haploid cells occurred at a slower rate than in diploid cells in sporulation medium. Diploid cells degraded 2.5% of the cellular protein/h in sporulation medium (34Betz H. Weiser U. Eur. J. Biochem. 1976; 70: 385-395Crossref PubMed Scopus (26) Google Scholar). Taken together, these results indicate that most proteins should not decrease below 70% of their original levels because of autophagy, even after 24 h of starvation. In wild-type cells, the amount of Ald6p was reduced to 18% of the initial level after 24 h of nitrogen starvation (Fig. 1B). This large decrease in Ald6p levels reflects preferential autophagic degradation. The result shown in Fig. 4C indicates that the specificity of Ald6p degradation may be achieved by a step of sequestration to the autophagosome. Suzuki et al. (22Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (794) Google Scholar, 31Suzuki K. Kamada Y. Ohsumi Y. Dev. Cell. 2002; 3: 815-824Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) indicated that the vacuolar targeting of the proform of API (via the Cvt pathway) required localization with the preautophagosomal structure, which plays a central role in autophagosome formation. In both Δatg11/cvt9 and Δatg19/cvt19 mutant cells, the proform of API localized to the cytosol away from the preautophagosomal structure and was not targeted to the vacuole (31Suzuki K. Kamada Y. Ohsumi Y. Dev. Cell. 2002; 3: 815-824Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 35Shintani T. Huang W.P. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2002; 3: 825-837Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). It was expected that Atg11p and Atg19p would be membrane receptors for the proform of API (24Kim J. Kamada Y. Stromhaug P.E. Guan J. Hefner-Gravink A. Baba M. Scott S.V. Ohsumi Y. Dunn Jr., W.A. Klionsky D.J. J. Cell Biol. 2001; 153: 381-396Crossref PubMed Scopus (216) Google Scholar, 25Scott S.V. Guan J. Hutchins M.U. Kim J. Klionsky D.J. Mol. Cell. 2001; 7: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). However, Ald6p degradation was not dependent on Atg11p (Fig. 1C, lanes 1, 2, 11, and 12) and Atg19p (data not shown). During nitrogen starvation, the half-life (t½) of Ald6p was 100 min (Fig. 2), and the half-life of API was 30 min (6Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (389) Google Scholar). These results indicate that Ald6p is not likely to be a cargo of the general Cvt pathway. One factor contributing to protein targeting is the existence of a membrane receptor; it is possible that the selective sequestration of Ald6p is mediated by a yet unknown molecule(s) on the autophagosome. Further studies of the molecular mechanisms underlying targeted autophagy are now in progress to investigate these possibilities. To address the physiological significance of this selective degradation, we analyzed the viability of Δald6 or ALD6 overexpressing cells. We have demonstrated that Ald6p enzymatic activity might be disadvantageous for the survival of yeast cells during nitrogen starvation (Fig. 5). Brejning and Jespersen (36Brejning J. Jespersen L. Int. J. Food Microbiol. 2002; 75: 27-38Crossref PubMed Scopus (36) Google Scholar) have previously reported that Ald6p levels increased during lag phase, the first hours after inoculation of the culture. Meaden et al. (13Meaden P.G. Dickinson F.M. Mifsud A. Tessier W. Westwater J. Bussey H. Midgley M. Yeast. 1997; 13: 1319-1327Crossref PubMed Scopus (74) Google Scholar) reported that the growth of Δald6 mutant cells is slower than that of wild-type cells in both YPD and synthetic medium. It is known that acetaldehyde dehydrogenase is closely related to lipid biosynthesis through the intermediary of acetyl-CoA synthase and fatty acid synthase in the cytosol. Because lipid biosynthesis is a critical process, the expression of Ald6p would be necessary during growth under nutrient conditions. Why is cytosolic Ald6p acetaldehyde dehydrogenase activity harmful under nitrogen starvation conditions? One possible explanations might be that Ald6p may disturb NADPH flux during nitrogen starvation. It is well known that glucose-6-phosphate dehydrogenase (Zwf1p; glucose-6-phosphate + NADP+ → 6-phosphogluconolactone + NADPH) is the greatest contributor to the reduction of NADP+ in the yeast cell. Grabowska and Chelstowska (37Grabowska D. Chelstowska A. J. Biol. Chem. 2003; 278: 13984-13988Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) have recently demonstrated that Δald6 Δzwf1 double mutant cells are not viable under normal growth conditions or under anaerobic growth conditions even in the presence of glutathione. It is suggested that Ald6p plays an important role in maintaining a high rate of NADPH/NADP+ cycling in the yeast cell. However, upon nitrogen starvation, both fatty acid and deoxyribonucleoside biosynthesis, which consume large amounts of NADPH, shut down immediately (38Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3704) Google Scholar, 39Murray R.K. Granner D.K. Mayes P.A. Rodwell V.M. Harper's Illustrated Biochemistry. 26th Ed. McGraw-Hill Co., Columbus, OH2003Google Scholar). We speculate that the reduction of NADP+ by Ald6p might be excessive in nitrogen-starved cells. An excessive amount of NADPH might inhibit the enzymatic activity of Zwf1p, which catalyzes the initial reaction of the pentose phosphate pathway. This pathway contributes to the synthesis of ribose-5-phosphate, which is an essential material for the generation of some amino acids and ribonucleotides (39Murray R.K. Granner D.K. Mayes P.A. Rodwell V.M. Harper's Illustrated Biochemistry. 26th Ed. McGraw-Hill Co., Columbus, OH2003Google Scholar). Ald4p, the mitochondrial acetaldehyde dehydrogenase, utilizes mainly NAD+ as a co-enzyme (14Tessier W.D. Meaden P.G. Dickinson F.M. Midgley M. FEMS Microbiol. Lett. 1998; 164: 29-34Crossref PubMed Google Scholar) and is induced during nitrogen starvation (38Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3704) Google Scholar). In our experiments, Δatg7 Δald4 mutant cells were not able to maintain high rates of viability like Δatg7 Δald6 cells (Fig. 5B). It is likely that the down-regulation of Ald6p by preferential autophagic degradation may optimize NADPH/NADP+ levels in the cytosol. Thus, Ald6p may have a bilateral character: it is beneficial in growth under nutrient conditions but disadvantageous to survival under nitrogen starvation. Here, we show that Ald6p is one example of a preferential substrate for autophagic degradation. Ald6p was the only major protein on the two-dimensional PAGE gel to decrease during starvation; however, it is still possible that other minor proteins behave like Ald6p. If we are able to find such proteins, it would help clarify the molecular mechanisms of selective autophagy and the physiological significance of the preferential degradation. We thank the Center for Analytical Instruments, National Institute for Basic Biology for technical assistance in MALDI-TOF mass spectrometry.
Referência(s)