Autophagy Is Required for Maintenance of Amino Acid Levels and Protein Synthesis under Nitrogen Starvation
2005; Elsevier BV; Volume: 280; Issue: 36 Linguagem: Inglês
10.1074/jbc.m506736200
ISSN1083-351X
AutoresJun Onodera, Yoshinori Ohsumi,
Tópico(s)Plant responses to water stress
ResumoAutophagy is a transport system of cytoplasmic components to the lysosome/vacuole for degradation well conserved in eukaryotes. Autophagy is strongly induced by nutrient starvation. Several specific proteins, including amino acid synthesis enzymes and vacuolar enzymes, are increased during nitrogen starvation in wild-type cells but not in autophagy-defective Δatg7 cells despite similar mRNA levels. We further examined deficiencies in these cells. Bulk protein synthesis was substantially reduced in Δatg7 cells under nitrogen starvation compared with wild-type cells. The total intracellular amino acid pool was reduced in Δatg7 cells, and the levels of several amino acids fell below critical values. In contrast, wild-type cells maintained amino acid levels compatible with life. Autophagy-defective cells fail to maintain physiologic amino acid levels, and their inability to synthesize new proteins may explain most phenotypes associated with autophagy mutants at least partly. Autophagy is a transport system of cytoplasmic components to the lysosome/vacuole for degradation well conserved in eukaryotes. Autophagy is strongly induced by nutrient starvation. Several specific proteins, including amino acid synthesis enzymes and vacuolar enzymes, are increased during nitrogen starvation in wild-type cells but not in autophagy-defective Δatg7 cells despite similar mRNA levels. We further examined deficiencies in these cells. Bulk protein synthesis was substantially reduced in Δatg7 cells under nitrogen starvation compared with wild-type cells. The total intracellular amino acid pool was reduced in Δatg7 cells, and the levels of several amino acids fell below critical values. In contrast, wild-type cells maintained amino acid levels compatible with life. Autophagy-defective cells fail to maintain physiologic amino acid levels, and their inability to synthesize new proteins may explain most phenotypes associated with autophagy mutants at least partly. In nature, organisms often encounter conditions in which nutrients are limiting, and they use an array of metabolic changes to survive through these periods. Under nitrogen starvation, cells mobilize stored nitrogen sources such as the vacuolar amino acid pool, express high affinity transporters to facilitate nitrogen uptake, and induce many enzymes controlling amino acid biosynthesis. Alterations in extracellular nutrients are typically most severe for unicellular organisms, and they have evolved mechanisms that respond to environmental changes more rapidly than multicellular organisms. Furthermore, when insufficient amounts of nitrogen are available, cells degrade their intracellular components to maintain essential cellular functions. In mammalian cells, most long lived cytoplasmic proteins are degraded in lysosomes/vacuoles (1Seglen P.O. Glaumann H. Ballard F.J. Lysosome: Their Role in Protein Breakdown. Academic Press, Orlando, FL1987: 371-414Google Scholar), and this process is called autophagy. In autophagy, a portion of the cytoplasm is sequestered into a double-membrane-bound structure, the autophagosome, which then fuses with the lysosome/vacuole, and its inner membrane and contents are degraded. We found that the yeast Saccharomyces cerevisiae induces autophagy under various nutrient starvation conditions (2Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (949) Google Scholar, 3Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (402) Google Scholar). A total of 16 ATG genes (ATG1–10, -12–14, and -16–18) essential for autophagosome formation were characterized in yeast (4Klionsky D.J. J. Cell Sci. 2005; 118: 7-18Crossref PubMed Scopus (759) Google Scholar). Most of these genes are well conserved in eukaryotes, and their identification facilitated many recent studies on the physiological significance of autophagy in higher eukaryotes (5Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3159) Google Scholar, 6Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2372) Google Scholar, 7Komatsu M. Waguri S. Ueno T. Iwata J. Murata S. Tanida I. Ezaki J. Mizushima N. Ohsumi Y. Uchiyama Y. Kominami E. Tanaka K. Chiba T. J. Cell Biol. 2005; 169: 425-434Crossref PubMed Scopus (1899) Google Scholar). Autophagy degraded cytoplasmic components including a significant amount of proteins, RNA in ribosomes, and phospholipids of organelle membranes (2Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (949) Google Scholar, 3Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (402) Google Scholar, 8Hamasaki M. Noda T. Baba M. Ohsumi Y. Traffic. 2005; 6: 56-65Crossref PubMed Scopus (140) Google Scholar). So far, the precise fate and the physiological importance of the recycling of each degradation product generated by autophagy are not known in particular. Recently, we examined changes in protein expression after long term nitrogen starvation and found Ald6p (NADP+-dependent cytosolic acetaldehyde dehydrogenase) is preferentially degraded via autophagy (9Onodera J. Ohsumi Y. J. Biol. Chem. 2004; 279: 16071-16076Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Additionally, it became apparent that heat shock proteins and enzymes involved in amino acid biosynthesis were up-regulated by nitrogen starvation in wild-type cells, but these changes did not occur in atg mutant cells. This observation could help clarify the physiological significance of autophagy in protein turnover. Here we show that protein synthesis in atg mutant cells is far lower than in wild-type cells under nitrogen starvation, and a decreased intracellular free amino acid pool limits protein synthesis in these cells. Yeast Strains, Media, and Culture—The S. cerevisiae strains used in this study were SEY6210 (MATα his3Δ200 leu2–3,112 lys2–801 trp1Δ901 ura3–52 suc2Δ9) (10Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (732) Google Scholar), JOY67 (SEY6210; Δatg7::kanMX4) (this study), X2180–1B (MATα SUC2 mal mel gal2 CUP1) (Yeast Genetic Stock Center), and JOY27 (X2180–1B; Δatg7::kanMX4) (this study). Standard techniques were used for yeast manipulation (11Bruke 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), SD medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, and 2% glucose), 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 2% glucose) was used. Yeast cells were grown in YPD medium to A600 = 1.0 at 30 °C and shifted to SD(-N) medium for the indicated times at 30 °C. Plasmid Construction—DNA manipulations were performed using standard methods (12Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual,2nd. Cold Spring HarborLaboratory, Cold Spring Harbor, NY1989Google Scholar). To create the expression vectors for Arg1p- or Hsp26p-3 × hemagglutinin (HA) 1The abbreviation used is: HA, hemagglutinin. epitope-tagged protein, novel XhoI and BamHI sites were added to each promoter region and the open reading frame lacking the stop codon (ARG1/YOL058w 2.2 kb and HSP26/YBR072w 1.6 kb) by genomic PCR amplification using each following primers: ARG1, 5′-CCGCTCGAGAGGTTGCCACATACATGGCCAAG-3′ and 5′-CGGGATCCCAAAGTCAACTCTTCACCTTTG-3′; HSP26, 5′-CCGCTCGAGCGTTGGACTTTTTTTAATATAAC-3′ and 5′-CGGGATCCGTTACCCCACGATTCTTGAGAAG-3′. Novel SpeI sites were added to the coding sequence of 3 × HA lacking the initiation codon (0.1 kb) by PCR amplification, and novel XbaI sites were added to the terminator sequence of ALD6/YPL061w (0.3 kb) by PCR amplification as described (9Onodera J. Ohsumi Y. J. Biol. Chem. 2004; 279: 16071-16076Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The resulting DNA fragments were cloned into the pRS316 centrometric plasmid (13Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to yield pJO128 (Arg1pHA) and pJO129 (Hsp26pHA). Immunoblot—Preparations of whole-cell lysates, SDS-PAGE and immunoblot analyses were performed as described previously (14Kirisako 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). Anti-HA epitope antibody (16B12) has purchased from Babco. Anti-alcohol dehydrogenase, anti-aminopeptidase I, and anti-carboxypeptidase Y antibodies have been described previously (3Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (402) Google Scholar, 15Hamasaki M. Noda T. Ohsumi Y. Cell Struct. Funct. 2003; 28: 49-54Crossref PubMed Scopus (79) Google Scholar, 16Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar). Northern Blot—Total RNA was isolated with the RNeasy mini kit (Qiagen). Total RNA (5 μg) was separated on 1% agarose containing 2.2 m formaldehyde by electrophoresis and transferred to BIODYNE A membrane (Pall). digoxigenin-labeled probes (0.5 kb) for ARG1, HSP26, and ACT1/YFL039c mRNA were prepared from each open reading frame fragment using PCR digoxigenin probe synthesis kit (Roche Applied Science). The mRNA-transferred membrane was incubated with each probe at 50 °C overnight, and signals were detected by anti-digoxigenin-AP (Roche Applied Science), CDP-Star (Roche Applied Science), and LAS-1000 system (Fuji Film). Whole-cell Amino Acid Analysis—Whole-cell free amino acid levels were quantitated as described previously (17Ohsumi Y. Kitamoto K. Anraku Y. J. Bacteriol. 1988; 170: 2676-2682Crossref PubMed Google Scholar, 18Kitamoto K. Yoshizawa K. Ohsumi Y. Anraku Y. J. Bacteriol. 1988; 170: 2683-2686Crossref PubMed Scopus (145) Google Scholar). Yeast cells (10 A600 units) were washed twice with distilled water, suspended in 500 μl of distilled water, and boiled for 15 min. The suspension was centrifuged for 3 min at 5,000 rpm, and the supernatant was collected. This extract was analyzed with an amino acid analyzer (Hitachi L-8500A). In Vivo Protein Synthesis Assay—Yeast cells (1 A600 unit) were suspended in fresh SD(-N) medium, and [14C]valine (Moravec MC277) was added at a final concentration of 10 μm (74 kBq/ml). Cultures were then incubated at 30 °C for 0, 2, and 4 min, and protein synthesis was stopped by adding 10 volumes of 11% w/v trichloroacetic acid solution (for trichloroacetic acid-insoluble fraction assay) or cold distilled water (for total cell assay). Trichloroacetic acid suspensions were incubated at 90 °C for 10 min and cooled to 4 °C, and precipitates were collected with membrane filters (MFTM-membrane filters, 0.45 μm for HA; Millipore). Cells in cold water were immediately collected with the filter. Incorporated radioactivity was determined in a liquid scintillation counter (Packard TRI-CARB 2700TR). Induction of Nitrogen Starvation-induced Proteins Requires Autophagy—Using two-dimensional PAGE analysis, we observed that the inability of cells to undergo autophagy blocked the up-regulation of several starvation-induced proteins. 2J. Onodera and Y. Ohsumi, unpublished observations. We chose two proteins for further analysis, Arg1p (argininosuccinate synthetase on arginine biosynthesis) and Hsp26p (heat shock protein of 26 kDa), whose mRNA levels are dramatically up-regulated within the initial 30 min of nitrogen starvation (19Gasch 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). Using immunoblot analysis, we quantified protein levels in wild-type (SEY6210) cells and autophagy-defective Δatg7 (JOY67) mutant cells under nitrogen starvation. In wild-type cells, Arg1pHA and Hsp26pHA levels increased by 10 and 40 fold after 24 h of starvation, respectively (Fig. 1A, lanes 1-5, B, and C). In contrast, protein up-regulation in Δatg7 mutant cells was substantially abrogated (Fig. 1A, lanes 6–10, B, and C). Two vacuolar proteinases, aminopeptidase I and carboxypeptidase Y, which are also known to be markedly induced under nitrogen starvation (19Gasch 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), behaved similarly (Fig. 1, A, D, and E). Levels of these proteins in Δatg7 cells were about 15–25% those seen in wild-type cells after 6 h of nitrogen starvation (Fig. 1, B–E). A similar defect was also observed for Δpep4, vacuolar proteinase A-defective cells (data not shown). These results clearly indicate that up-regulation of these proteins under nitrogen starvation requires autophagic degradation. Since it is possible that protein up-regulation is controlled at the level of gene transcription, we examined ARG1 and HSP26 mRNA levels by Northern blot. Interestingly, in both wild-type (SEY6210) and Δatg7 (JOY67) cells, ARG1 and HSP26 mRNA levels increased dramatically in response to starvation and reached a maximum level within 3 h of starvation (Fig. 2). Even after 12 h of starvation, mRNA levels were elevated compared with growing cells (Fig. 2). These data clearly demonstrate that autophagy-defective cells are able to induce and maintain expression of mRNA encoding starvation-induced genes as well as wild-type cells, which means nucleotide pool does not limit transcription under nitrogen starvation. The decreased protein levels in these cells are due to a post-transcriptional defect. Bulk Protein Synthesis Requires Autophagy under Nitrogen Starvation—Next, we assessed in vivo protein synthesis using [14C]valine under nitrogen starvation. Amino acid uptake was measured by total cellular [14C]valine levels, and protein synthesis was estimated by the degree of [14C]valine incorporation in the 10% w/v trichloroacetic acid-insoluble fraction. We confirmed these assumptions using cycloheximide (25 μg/ml). In the presence of cycloheximide, [14C]valine uptake was not inhibited, and no radioactivity was detectable in trichloroacetic acid-precipitated material (data not shown). We measured total [14C]valine uptake (Fig. 3A) and incorporation into proteins (Fig. 3B) as well as total valine levels by amino acid analysis (Fig. 3C). Δatg7 cells (gray bars) had greater [14C]valine uptake than wild-type cells (black bars) (Fig. 3A), and this may be due to the overall decreased amino acid levels seen in these cells (Fig. 3C). The degree of protein synthesis was estimated from [14C]valine incorporated in protein and the initial specific activity calculated with cold and radioactive valine contents. Protein synthesis in both wild-type (X2180–1B) and Δatg7 (JOY27) cells was dramatically reduced for the initial 3 h of starvation. After 3 h of nitrogen starvation, however, protein synthesis was restored in wild-type cells (black bars), but Δatg7 cells remained biosynthetically less active (gray bars). The delay in Arg1p, Hsp26p, and carboxypeptidase Y expression correlates with this 3 h period of decreased protein synthesis (Fig. 1, B–D). After 24 h of starvation, total protein synthesis in Δatg7 cells was 17% of wild-type cells (Fig. 3D). This value corresponds to the relative expression levels observed for individual starvationinduced protein in Δatg7 cells compared with wild-type cells (15–25%; Fig. 1). Therefore, the failure of induction of Arg1p and Hsp26p in Δatg7 cells is not specific event, but due to decline of general protein synthesis. We evaluate that protein synthesis under nitrogen starvation requires autophagy, and the products of autophagic degradation are essential for protein synthesis. Autophagy Contributes to the Maintenance of Free Amino Acid Pool—Mutant cells deficient in autophagy cannot survive under long-term nitrogen starvation (20Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1390) Google Scholar) or sulfur starvation.2 Taken together with the data presented above, we hypothesized that amino acids generated during autophagy were a limiting factor for protein synthesis under starvation conditions. Total amino acid level of wild-type cells decreased dramatically during the first 2 h of starvation. Since autophagic activity measured by the alkaline phosphatase (Pho8Δ60p) assay method shows an apparent lag time of 1 h (16Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar), in the early phase of nitrogen starvation consumption of amino acids must far exceed its supply via autophagy, resulting in an abrupt reduction of amino acid pools. But the amino acid level was partly restored during 3–6 h of starvation, and the level of 40.6 nmol/A600 unit was seen after 12 h of starvation (Fig. 4A; Supplemental data, Table SI). The size of the amino acid pool closely corresponded to that of X2180–1B cells growing in amino acid-free synthetic medium (SD) (44 nmol/A600 unit cells). In contrast, the total amino acid levels in Δatg7 cells (JOY27) continued to decrease dramatically during nitrogen starvation throughout the starvation and reached 9.4 nmol/A600 unit cells after 24 h of starvation (Fig. 4A; supplemental Table SI). Other autophagy-defective mutants, atg1 and atg2 cells, also failed to maintain the free amino acid pool under nitrogen starvation (data not shown). Several amino acid levels, such as histidine, methionine, and glutamine/glutamate, of Δatg7 cells fell into much lower levels than those of wild-type cells (Fig. 4, B–D). Exceptionally, the proline content of Δatg7 cells was similar to wild-type cells throughout the starvation period (Fig. 4E). The cysteine content of wild-type cells was dramatically increased at 3 h of starvation (Fig. 4F), and we will discuss about the physiological significance of this finding below. Autophagy appears essential for the maintenance of free amino acid levels during starvation conditions. In the absence of autophagy, several amino acid levels fall below a critical value, and cells may show reduced bulk protein synthesis during nitrogen starvation. Free Amino Acid Contents Limit Protein Synthesis—Decreased protein synthesis in autophagy-defective cells may be due to translation inhibition. When we added [14C]valine to cultures of 6-h starved cells, protein incorporation of [14C]valine occurred with similar kinetics in both Δatg7 cells and wild-type cells (Fig. 5A), suggesting that the function of translational machinery (e.g. ribosome) in Δatg7 cells is normal. To further demonstrate that protein synthesis is determined by free amino acid levels, we modified the protein synthesis assay somewhat. Nitrogen-starved cells were preincubated in SD + CA medium containing an excess of free amino acids for 5 min at 30 °C, washed with SD(-N) twice, and then cells were immediately subjected to the assay of protein synthesis using [14C]valine. Following incubation in complete medium, the amino acid pool of Δatg7 cells (JOY27) was restored (Fig. 5B, gray and white bars), and protein synthesis occurred comparable with wild-type cells (X2180-1B) (Fig. 5C, dark gray and white bars). Thus, under nitrogen starvation protein synthesis is limited by the size of the free amino acid pool. We previously observed differences in protein expression in autophagy-defective cells, and we investigated these differences in greater detail. Bulk protein synthesis in Δatg7 cells was dramatically lower than that seen in wild-type cells under nitrogen depletion. Furthermore, Δatg7 cells could not maintain intracellular amino acid levels; in several hours after shift to starvation, amino acid levels fell below critical values. Kuma et al. (6Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2372) Google Scholar) recently reported that Atg5 knock-out mice have reduced plasma and tissue amino acid levels compared with wild-type mice under fasting conditions; however, the reduction was partial (about 20–30% reduction) in tissue of mammals. Our results clearly demonstrated that in yeast the free amino acid pools change more dramatically and become a limiting factor for protein synthesis during cellular starvation without autophagy. As shown in Fig. 4, A and F, after 3 h of starvation, the amino acid levels of wild-type cells were abnormal with cysteine representing 75% of the total amino acids. The reduced protein synthesis observed at 3 h likely arises from the depletion of other amino acids (Fig. 3), and this was confirmed by the result that cells nitrogen-starved for 3 h retained high protein synthesis activity when supplied with exogenous amino acids (Fig. 5). Cysteine content rapidly diminished after 3 h of starvation, and the other amino acids achieved normal representation (Fig. 4F). Protein synthesis was also restored at this time (Fig. 3). Thus, the total amino acid content is less important for protein synthesis than the critical levels of each amino acid. Enzymes involved in both cysteine biosynthesis and sulfur metabolism are highly expressed during the initial 3-h nitrogen starvation (19Gasch 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), and sulfur metabolism consumes a large amount of NADPH (21Thomas D. Surdin-Kerjan Y. Microbiol. Mol. Biol. Rev. 1997; 61: 503-532Crossref PubMed Scopus (530) Google Scholar). We previously hypothesized that Ald6p is preferentially degraded (9Onodera J. Ohsumi Y. J. Biol. Chem. 2004; 279: 16071-16076Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) during nitrogen starvation because it is a potent NADPH production enzyme (22Grabowska D. Chelstowska A. J. Biol. Chem. 2003; 278: 13984-13988Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and its degradation may reduce NADPH levels under nitrogen starvation. However, this preferential degradation is not enough to eliminate NADPH in the early phase of nitrogen depletion (9Onodera J. Ohsumi Y. J. Biol. Chem. 2004; 279: 16071-16076Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Sulfur metabolism may further reduce NADPH levels during the early stages of nitrogen starvation. The accumulation of cysteine is the natural cellular end point of sulfur metabolism. Zubenko and Jones (23Zubenko G.S. Jones E.W. Genetics. 1981; 97: 45-64Crossref PubMed Google Scholar) presented that vacuolar proteinase A mutant (pep4-3) was defective in starvation-induced protein degradation and spore formation. Tsukada and Ohsumi (20Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1390) Google Scholar) showed that autophagy is essential for spore formation, and our data suggest that the decreased synthesis of proteins required for meiosis and spore formation may give rise to this phenotype in autophagy-deficient cells (24Kupiec M. Byers B. Esposito R.E. Mitchell A.P. Pringle J.R. Broach J.R. Jones E.W. Cell Cycle and Cell Biology, The Molecular and Cellular Biology of the Yeast Saccharomyces. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 889-1036Google Scholar). Recent studies of ATG gene knock-out organisms indicate that autophagy is essential for stress-induced differentiation and development; autophagy mutants of Dictyostelium discoideum exhibit defective fruiting body formation (25Otto 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, 26Otto G.P. Wu M.Y. Kazgan N. Anderson O.R. Kessin R.H. J. Biol. Chem. 2004; 279: 15621-15629Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), Caenorhabditis elegans mutants have defective dauer development (27Melendez A. Talloczy Z. Seaman M. Eskelinen E.L. Hall D.H. Levine B. Science. 2003; 301: 1387-1391Crossref PubMed Scopus (1017) Google Scholar), and mutant Drosophila melanogaster experience premature death at or after the third larval to pupal stages (28Scott R.C. Schuldiner O. Neufeld T.P. Dev. Cell. 2004; 7: 167-178Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar). These developmental defects may also be caused by reduced synthesis of proteins required for intracellular remodeling. Here we clearly demonstrated this new novel phenotype, reduced protein synthesis, for autophagy-defective cells under nitrogen starvation. Atg5 knock-out mice showed that deficiencies in branched-chain amino acids could cause substantial tissue energy deficits under fasting condition (6Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2372) Google Scholar). Although we also identified a decrease in branched-chain amino acid levels in yeast autophagy mutant (supplemental Table SI), S. cerevisiae has neither branched-chain α-ketoacid dehydrogenase nor dihydrolipoamide branched-chain transacylase within the branched-chain amino acid degradation pathway. Thus, free amino acids should not be important as an energy source for yeast cells. In our experiments, sufficient glucose was present; therefore, amino acids may be re-utilized directly for protein synthesis. In Escherichia coli, the Lon protease is activated by starvation-induced polyphosphate, and it degrades ribosomal proteins to provide amino acids under starvation conditions (29Kuroda A. Nomura K. Ohtomo R. Kato J. Ikeda T. Takiguchi N. Ohtake H. Kornberg A. Science. 2001; 293: 705-708Crossref PubMed Scopus (293) Google Scholar). The reduced protein synthesis observed in Δatg7 cells is not caused by an irreversible inactivation of translational machinery (Fig. 5), but the critical levels of several amino acids should result in uncharged forms of tRNA. The ubiquitin/proteasome system is also involved in protein turnover and degradation. At least in yeast, our results clearly showed that amino acids generated by the ubiquitin/proteasome system are not sufficient for the maintenance of the amino acid pool under nitrogen depletion (Fig. 4). One A600 unit of yeast cells contains about 150–200 μg of total protein (data not shown). After 6 h nitrogen starvation, wild-type cells contain 10 μg of amino acids/A600 unit cells (equivalent to about 77 nmol of amino acids/A600 unit cells; supplemental Table SI); therefore, overall levels are dynamically maintained. Previous biochemical and morphological studies have indicated that autophagy degrades around 2–4% of the total proteins/h in yeast (2Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (949) Google Scholar, 3Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (402) Google Scholar, 30Scott 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); therefore, it is likely that intracellular amino acids are primarily supplied by autophagic degradation. In this study, we demonstrated that protein synthesis during starvation is dramatically affected by the intracellular amino acid level, and an inability to undergo protein synthesis may partly explain the loss of viability phenotype of autophagy-deficient cells under nitrogen starvation. This provides new insight into the physiological significance of autophagy in maintaining homeostasis. Amino acid pools reduce the protein synthesis as a whole; however, to adapt nutrient starvation stress, cells must induce the synthesis of several sets of proteins, whose synthesis must be affected more severely than that of constitutively synthesized proteins. We are grateful to Shigemi Takami and Tomoko Mori (Center for Analytical Instruments, National Institute for Basic Biology) and Hideko Yomo (Suntory Ltd.) for their assistance with the amino acid analyses. Download .pdf (.03 MB) Help with pdf files
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