Key points are not available for this paper at this time.
Mutation in the Saccharomyces cerevisiae APG14 gene causes a defect in autophagy. Cloning and structural analysis of the APG14 gene revealed that APG14 encodes a novel hydrophilic protein with a predicted molecular mass of 40.5 kDa, and that Apg14p has a coiled-coil motif at its N terminus region. We found that overproduction of Apg14p partially reversed the defect in autophagy induced by theapg6-1 mutation. The apg6-1 mutant was found to be defective not only in autophagy but also in sorting of carboxypeptidase Y (CPY), a vacuolar-soluble hydrolase, to the vacuole. However, overexpression of APG14 did not alter the CPY sorting defect of the apg6-1 mutant, nor did theapg14 null mutation affect the CPY sorting pathway. Structural analysis of APG6 revealed that APG6is identical to VPS30, which is involved in a retrieval step of the CPY receptor, Vps10p, to the late-Golgi from the endosome (Seaman, M. N. J., Marcusson, E. G., Cereghino, J. L., and Emr, S. D. (1997) J. Cell Biol. 137, 79–92). Subcellular fractionation indicated that Apg14p and Apg6p peripherally associated with a membrane structure(s). Apg14p was co-immunoprecipitated with Apg6p, suggesting that they form a stable protein complex. These results imply that Apg6/Vps30p has two distinct functions in the autophagic process and the vacuolar protein sorting pathway. Apg14p may be a component specifically required for the function of Apg6/Vps30p through the autophagic pathway. Mutation in the Saccharomyces cerevisiae APG14 gene causes a defect in autophagy. Cloning and structural analysis of the APG14 gene revealed that APG14 encodes a novel hydrophilic protein with a predicted molecular mass of 40.5 kDa, and that Apg14p has a coiled-coil motif at its N terminus region. We found that overproduction of Apg14p partially reversed the defect in autophagy induced by theapg6-1 mutation. The apg6-1 mutant was found to be defective not only in autophagy but also in sorting of carboxypeptidase Y (CPY), a vacuolar-soluble hydrolase, to the vacuole. However, overexpression of APG14 did not alter the CPY sorting defect of the apg6-1 mutant, nor did theapg14 null mutation affect the CPY sorting pathway. Structural analysis of APG6 revealed that APG6is identical to VPS30, which is involved in a retrieval step of the CPY receptor, Vps10p, to the late-Golgi from the endosome (Seaman, M. N. J., Marcusson, E. G., Cereghino, J. L., and Emr, S. D. (1997) J. Cell Biol. 137, 79–92). Subcellular fractionation indicated that Apg14p and Apg6p peripherally associated with a membrane structure(s). Apg14p was co-immunoprecipitated with Apg6p, suggesting that they form a stable protein complex. These results imply that Apg6/Vps30p has two distinct functions in the autophagic process and the vacuolar protein sorting pathway. Apg14p may be a component specifically required for the function of Apg6/Vps30p through the autophagic pathway. Cell growth is governed by a fine-tuned balance between the synthesis and degradation of proteins. In mammalian cells, both lysosomal and nonlysosomal protein degradation mechanisms are responsible for turnover of endogenous proteins. Intracellular proteolytic activity is essential for cells to survive in various extracellular environments. Autophagy is the bulk degradation of cytoplasmic components in the lysosome/vacuole (1Dunn Jr., W.A. J. Cell Biol. 1990; 110: 1923-1933Crossref PubMed Scopus (515) Google Scholar, 2Dunn Jr., W.A. J. Cell Biol. 1990; 110: 1935-1945Crossref PubMed Scopus (371) Google Scholar, 3Kopitz J. Kisen G.O. Gordon P.B. Bohley P. Seglen P.O. J. Cell Biol. 1990; 111: 941-953Crossref PubMed Scopus (191) Google Scholar, 4Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (969) Google Scholar). Under serum starvation conditions, animal cells induce autophagy to supply amino acids (5Mortimore G.E. Schworer C.M. Nature. 1977; 270: 174-176Crossref PubMed Scopus (252) Google Scholar). Autophagy starts with formation of the autophagosome, a cytoplasmic membrane structure surrounding cytosolic components or organelles. The outer membrane of the autophagosome subsequently fuses with the lysosomal membrane, and the contents are degraded in a lysosomal proteinase-dependent manner (6Seglen P.O. Glamann H. Ericsson J.L. Marzella L. Lysosomes. Academic Press, London1987: 371-414Google Scholar). Although mammalian autophagy has been characterized with morphological and biochemical approaches, the molecular basis of each process is still unclear. Recent studies revealed that autophagy takes place in the budding yeast, Saccharomyces cerevisiae, in a similar manner to that of higher eucaryotes (7Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (404) Google Scholar, 8Baba M. Osumi M. Ohsumi Y. Cell Struct. Funct. 1995; 20: 465-471Crossref PubMed Scopus (128) Google Scholar). Several lines of investigation showed that yeast autophagy is composed of the processes as follows: (i) starvation signaling, (ii) formation of autophagosome, (iii) targeting of autophagosome to the vacuole, (iv) fusion of the outer membrane of the autophagosome to the vacuolar membrane and release of the autophagic body in the vacuole, and (v) degradation of the autophagic body in the vacuole (4Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (969) Google Scholar, 7Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (404) Google Scholar, 8Baba M. Osumi M. Ohsumi Y. Cell Struct. Funct. 1995; 20: 465-471Crossref PubMed Scopus (128) Google Scholar, 9Noda T. Ohsumi Y. J. Biol. Chem. 1998; 273: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar). To elucidate the complex phenomena at a molecular level, autophagy-defective mutants (apg mutants) were isolated, and at least 14 APG genes were shown to be essential for yeast autophagy (10Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1427) Google Scholar). The phenotypic similarities among the 14 apg mutants suggest that the Apg proteins are involved in close processes in the autophagic pathway. Previous morphological studies showed that none of the apg mutants could form autophagosomes, 1M. Baba, unpublished data.suggesting the APG products function at autophagosome formation or at earlier steps of autophagic processes. So far, most of the APG genes have been cloned and characterization of the Apg proteins is underway (11Kametaka S. Matsuura A. Wada Y. Ohsumi Y. Gene ( Amst. ). 1996; 178: 139-143Crossref PubMed Scopus (93) Google Scholar, 12Funakoshi T. Matsuura A. Noda T. Ohsumi Y. Gene ( Amst. ). 1997; 192: 207-213Crossref PubMed Scopus (140) Google Scholar, 13Matsuura A. Tsukada M. Wada Y. Ohsumi Y. Gene ( Amst. ). 1997; 192: 245-250Crossref PubMed Scopus (390) Google Scholar). However, their functions in the autophagic pathway remain to be elucidated. In this study, we report the structural and functional analyses of Apg14p and Apg6p. APG6 were identical to VPS30, which is involved in the vacuolar protein sorting pathway (14Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (739) Google Scholar, 15Seaman M.N. Marcusson E.G. Cereghino J.L. Emr S.D. J. Cell Biol. 1997; 137: 79-92Crossref PubMed Scopus (324) Google Scholar). Our findings suggest that Apg6/Vps30p has two distinct functions, both on autophagy and vacuolar protein sorting pathways, and that Apg14p is specifically required for the function of Apg6/Vps30p in autophagy. Yeast strains used (Table I) were derived from X2180-1A and X2180-1B (Yeast Genetic Stock Center, Berkeley, CA). The media used for yeast were described previously (11Kametaka S. Matsuura A. Wada Y. Ohsumi Y. Gene ( Amst. ). 1996; 178: 139-143Crossref PubMed Scopus (93) Google Scholar). Standard genetic methods were performed as described previously (16Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar). Yeast transformation was carried out as described elsewhere (17Gietz R.D. Schiestl R.H. Willems A.R. Woods R.A. Yeast. 1995; 11: 355-360Crossref PubMed Scopus (1712) Google Scholar).Table IYeast strains, genotypes, and sourceStrainsGenotypesSourceYW5–1BMAT a leu2 ura3 trp1Y. WadaYNY1MAT a/ MATα leu2/leu2 ura3/ura3 trp1/trp1Y. WadaTN124MAT a ura3 trp1 leu2 PHO8::PHO8 Δ60 Δpho13::LEU2Nodaet al. (24Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar)M19–4-4MAT a apg6–1 ura3M. TsukadaMT37 4–3MAT a apg5–1 ura3M. IsukadaSKY6DPMAT a apg6–1 ura3 PHO8::PHO8 Δ60This workSKD6DPMAT a Δapg6::LEU2 ura3 leu2 trp1 PHO8::PHO8 Δ60This workSKD6–1DMAT a Δapg6::LEU2 ura3 leu2 trp1This workMT54–4-2MAT a apg14–1 ura3M. TsukadaSKD14–1OMAT a Δapg14::LEU2 ura3 leu2 trp1This workSKD14DPMAT a Δapg14::LEU2 ura3 leu2 trp1 PHO8::PHO8 Δ60This workSKDV29LMAT a Δvps29::LEU2 ura3 leu2 trp1This workSKDV35LMAT a Δvps35::LEU2 ura3 leu2 trp1This work Open table in a new tab Assay of alkaline phosphatase was performed as described previously (9Noda T. Ohsumi Y. J. Biol. Chem. 1998; 273: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar). Protein concentration of the cell lysate was examined with Bradford's method (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Standard molecular biological techniques for the manipulation of DNA were used throughout this study (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Yeast strain MT54-4-2 was transformed with a yeast genomic library based on YCp50. Approximately 3,200 Ura+ transformants were replica-plated to 0.17% yeast nitrogen base without amino acids and ammonium sulfate and 2% glucose (SD(−N)) plates including 5 μg/ml phloxine B and incubated for 48–72 h at 30 °C. were and subsequently examined for their autophagic activity by a morphological as described previously (4Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (969) Google Scholar, M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1427) Google Scholar). were and the genomic were from analysis showed that the genomic was identical to a on and the a was found to the The DNA was Hieter P. PubMed Google and subsequently at the APG14 not that the APG14 the in was with and with the from J.S. L. Yeast. 1990; PubMed Scopus Google Scholar). The was with and and a strain and were of the was by not was and were and on Cloning of the APG6 gene was performed as described that we used a yeast genomic library based on the and we the that the the was at the were by with and S. Res. 1990; PubMed Scopus Google Scholar). The DNA was by and used for for DNA To the APG6 mutant, the was cloned at and the was with the from The was and the was used for of the APG6 The was as described previously Methods PubMed Scopus Google Scholar). In genomic DNA was from mutant strain as described elsewhere (16Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google and the mutant was by a and with DNA The cells were with both the DNA of and which is the in the APG6 transformants were examined for Apg by for the of used the DNA was and the mutation was autophagic body carboxypeptidase Y The of APG6 was to a The fusion protein was and with from The fusion protein was used for of in a was carried out at yeast lysate was by cells with as described previously N. A. Ohsumi Y. Wada Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). analyses were performed as described previously (24Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar, N. A. Ohsumi Y. Wada Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). sorting of carboxypeptidase Y was by the method previously M.N. Marcusson E.G. Cereghino J.L. Emr S.D. J. Cell Biol. 1997; 137: 79-92Crossref PubMed Scopus (324) Google Scholar). of CPY and in the and and were as described previously Y. K. T. K. Y. Mol. Cell. Biol. 1990; PubMed Scopus Google Scholar, K. Noda T. Ohsumi Y. Cell Struct. Funct. 1997; PubMed Scopus Google Scholar). Subcellular fractionation by was performed as described elsewhere N. A. Ohsumi Y. Wada Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google with media yeast 2% for cells and for cell were in μg/ml μg/ml and μg/ml at the cell lysate was subsequently at for to a and the was at for h to a and was to and was carried out as described the cell were as described and with and 2% on for 30 and at for The and were to of Apg6p was performed as described elsewhere Y. H. Y. Y. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google with In yeast cells were with to and in 5 5 and of cell and by at the protein concentration was The was to a protein concentration of with To the of and of protein were and was performed at for To the protein the was at for and the was the was of of lysate was used for of to the lysate and incubated at for of protein were and incubated at for h to the and with and the proteins were by the protein with for 5 To DNA and the gene were from genomic DNA with and of the was the was with To a of the genomic was to the was with The were cells to the genomic of the genomic was with a genomic we the mutant which is defective in autophagy (10Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1427) Google Scholar). cells were incubated in a the membrane structure of are in the vacuole However, mutant in the vacuole, that the mutation causes a defect in autophagy. the mutant showed of nitrogen of the mutant cells nitrogen starvation for of cells were still To the APG14 gene the gene was cloned by of the defect in of starvation as described previously (11Kametaka S. Matsuura A. Wada Y. Ohsumi Y. Gene ( Amst. ). 1996; 178: 139-143Crossref PubMed Scopus (93) Google Scholar). of APG14 showed that encodes a hydrophilic protein of amino acids with a predicted molecular mass of 40.5 The between amino and of Apg14p that are predicted to coiled-coil A. M. Stock J. PubMed Scopus Google Scholar). base revealed that Apg14p is a novel protein to protein at To the phenotypic of a of Apg14p a null mutant of was by the of APG14 with gene The null mutant was and showed growth on APG14 function is not required for growth of the cell not autophagic activity of the null mutant was with morphological and biochemical shown in was defective in of in the vacuole nitrogen starvation and was also defective in alkaline phosphatase (24Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar). These results that APG14 is essential for autophagy. To the gene of of Apg14p was by the amino acids of were the N terminus of Apg14p to a for characterization of A molecular mass of the Apg14p was A the defect in autophagy of the the Apg14p was functional in not Mol. Cell. Biol. PubMed Scopus Google which the a protein was in yeast cell by The was found to be in cells and the of the did not The of was by fractionation We found that most of was in in the To the of the in the were shown in was with and partially with or that with a membrane and the be Apg14p not have or for The similar by mutants that of the Apg proteins in the autophagic processes. was found to peripherally with a membrane, suggesting are components which with To we for genetic between APG14 and APG We found that apg6-1 cells a APG14 of the in the vacuole The that the autophagic defect of the apg6-1 mutant was with APG14 the was not with APG6 overexpression not characterization of the APG6 of was carried out APG6 encodes a protein with molecular mass of and has a of The of Apg6p is predicted to form coiled-coil A. M. Stock J. PubMed Scopus Google Scholar). including for or for were To the mutation of theapg6-1 the mutant was cloned from mutant genomic DNA and was carried out Structural analysis of the apg6-1 mutant revealed that a to at base a mutation at in the apg6-1 mutant to the of the genomic APG6 was as described The strain was that Apg6p function is not essential for growth in cells showed a defect in autophagy and of nitrogen starvation similar to the not To the of the mutants on autophagic and were transformed with activity of the apg6-1 mutant was by overexpression of However, the was not in the 5 analysis showed that theapg6-1 mutant gene was as a protein of not These results suggest that the of the form of Apg6p is for of autophagy by Apg14p overproduction of the mutant Apg6p from the apg6-1 the autophagic defect of the not suggesting the of the protein essential function for Apg6p. To gene we a Apg6p The a protein in the lysate of the protein was not in the mutant, we that this protein is the Subcellular of Apg6p was by fractionation a Apg6p was in and was in the shown in Apg6p was by with the with or Apg6p. that Apg6p peripherally with membrane structure(s). and mutants have similar and overproduction of Apg14p the autophagic defect of theapg6-1 mutant Apg14p and Apg6p to be associated with membrane and These results suggest that Apg14p and Apg6p and through autophagic processes. To this of Apg6p was carried out from cells were and the was to with The were used for analysis with shown in was with is and Apg6p To the between Apg14p and Apg6p is by membrane the cell lysate was with We found that was also co-immunoprecipitated with Apg6p the cell been with suggesting that Apg6p and Apg14p this was in both and Structural analysis of APG6 revealed that is identical to the which is involved in sorting the vacuolar was cloned and characterized M.N. Marcusson E.G. Cereghino J.L. Emr S.D. J. Cell Biol. 1997; 137: 79-92Crossref PubMed Scopus (324) Google Scholar, Mol. Biol. Cell. 1992; PubMed Scopus Google Scholar). mutant cells, was shown to be to vacuole M.N. Marcusson E.G. Cereghino J.L. Emr S.D. J. Cell Biol. 1997; 137: 79-92Crossref PubMed Scopus (324) Google Scholar, E.G. Cereghino J.L. E. Emr S.D. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). is to be a CPY and required for of CPY to the endosome from the Emr and a that is involved in the retrieval process of the CPY from the endosome to late-Golgi M.N. Marcusson E.G. Cereghino J.L. Emr S.D. J. Cell Biol. 1997; 137: 79-92Crossref PubMed Scopus (324) Google Scholar). To the apg6-1 mutant a CPY we performed a shown in cells showed of the form of CPY to the extracellular that the apg6-1 mutant is defective in CPY Apg14p a stable protein complex with was also to be involved in the CPY sorting pathway. To we examined the CPY We found that the cells showed sorting of CPY that Apg14p function is not required for the CPY sorting pathway. shown APG14 overexpression the autophagic defect of the apg6-1 To also the CPY sorting defect in theapg6-1 mutant, and of CPY were shown in of CPY was in the apg6-1 mutant suggesting that APG14 overexpression the CPY sorting defect of the defect in autophagy. previously and are to function through the vacuolar protein pathway at close steps M.N. Marcusson E.G. Cereghino J.L. Emr S.D. J. Cell Biol. 1997; 137: 79-92Crossref PubMed Scopus (324) Google Emr S.D. Mol. Biol. Cell. 1992; PubMed Scopus Google Scholar). To the that the was from its defect in the vacuolar protein sorting we examined the and in autophagy. shown in mutants in their as in cells, the did not that genes are not essential for the autophagic process and the autophagic defect in mutant is not by of the CPY and CPY a of autophagy defective mutants (apg mutants) was and 14 were shown to be involved in autophagy in yeast (10Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1427) Google Scholar). Under starvation conditions, the apg mutants in for bulk protein degradation and (10Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1427) Google Scholar). analyses of apg mutants showed that none of autophagosomes, suggesting that the Apg proteins are involved in steps of as the formation of the In this we characterized a novel which is essential for autophagy. Apg14p is a hydrophilic protein with coiled-coil at its N the autophagic defect of theapg6-1 Structural analysis revealed is identical to VPS30, which is involved in the vacuolar protein sorting pathway M.N. Marcusson E.G. Cereghino J.L. Emr S.D. J. Cell Biol. 1997; 137: 79-92Crossref PubMed Scopus (324) Google Scholar). APG6 encodes a hydrophilic protein with a Apg14p and Apg6p form a protein and they are associated in However, their is still unclear. Apg14p and Apg6p form a protein is that they function in the autophagic pathway. A of this complex is peripherally associated with membrane structure(s). However, a showed that Apg14p was not with most of Apg6p was Apg14p and Apg6p were still in in and T. M. and Y. unpublished These that components are in the protein complex. A report on Apg6/Vps30p showed that this protein is associated with membrane, most was in the M.N. Marcusson E.G. Cereghino J.L. Emr S.D. J. Cell Biol. 1997; 137: 79-92Crossref PubMed Scopus (324) Google Scholar). 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Kametaka et al. (Sat,) studied this question.
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