Search tips
Search criteria 


Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. 1997 June; 179(12): 3875–3883.
PMCID: PMC179195

AUT3, a serine/threonine kinase gene, is essential for autophagocytosis in Saccharomyces cerevisiae.


Autophagocytosis is a starvation-induced process, carrying proteins destined for degradation to the lysosome. In the yeast Saccharomyces cerevisiae, the autophagic process is visualized by the appearance of autophagic vesicles in the vacuoles of proteinase yscB-deficient strains during starvation. aut3-1 mutant cells which exhibit a block in the autophagic process have been isolated previously. By using the drastically reduced sporulation frequency of homozygous aut3-1 diploid cells, the AUT3 gene was cloned by complementation. The Aut3 protein consists of 897 amino acids. The amino-terminal part of the protein shows significant homologies to serine/threonine kinases. aut3 null mutant cells are fully viable on rich media but show a reduced survival rate upon starvation. They are unable to accumulate autophagic vesicles in the vacuole during starvation. Starvation-induced vacuolar protein breakdown is almost completely impaired in aut3-deficient cells. Vacuolar morphology and acidification are not influenced in aut3-deficient cells. Also, secretion of invertase, endocytic uptake of Lucifer Yellow, and vacuolar protein sorting appear wild type like in aut3-deficient cells, suggesting autophagocytosis as a novel route for the transport of proteins from the cytosol to the vacuole. By using a fusion of Aut3p with green-fluorescent protein, Aut3p was localized to the cytosol.

Full Text

The Full Text of this article is available as a PDF (14M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ammerer G, Hunter CP, Rothman JH, Saari GC, Valls LA, Stevens TH. PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors. Mol Cell Biol. 1986 Jul;6(7):2490–2499. [PMC free article] [PubMed]
  • Baba M, Takeshige K, Baba N, Ohsumi Y. Ultrastructural analysis of the autophagic process in yeast: detection of autophagosomes and their characterization. J Cell Biol. 1994 Mar;124(6):903–913. [PMC free article] [PubMed]
  • Blommaart EF, Luiken JJ, Blommaart PJ, van Woerkom GM, Meijer AJ. Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. J Biol Chem. 1995 Feb 3;270(5):2320–2326. [PubMed]
  • Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802–805. [PubMed]
  • Conibear E, Stevens TH. Vacuolar biogenesis in yeast: sorting out the sorting proteins. Cell. 1995 Nov 17;83(4):513–516. [PubMed]
  • Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY. Understanding, improving and using green fluorescent proteins. Trends Biochem Sci. 1995 Nov;20(11):448–455. [PubMed]
  • Cuervo AM, Dice JF. A receptor for the selective uptake and degradation of proteins by lysosomes. Science. 1996 Jul 26;273(5274):501–503. [PubMed]
  • Cyr DM, Ungermann C, Neupert W. Analysis of mitochondrial protein import pathway in Saccharomyces cerevisiae with translocation intermediates. Methods Enzymol. 1995;260:241–252. [PubMed]
  • Dice JF. Peptide sequences that target cytosolic proteins for lysosomal proteolysis. Trends Biochem Sci. 1990 Aug;15(8):305–309. [PubMed]
  • Dulic V, Egerton M, Elguindi I, Raths S, Singer B, Riezman H. Yeast endocytosis assays. Methods Enzymol. 1991;194:697–710. [PubMed]
  • Dunn WA., Jr Studies on the mechanisms of autophagy: formation of the autophagic vacuole. J Cell Biol. 1990 Jun;110(6):1923–1933. [PMC free article] [PubMed]
  • Dunn WA., Jr Studies on the mechanisms of autophagy: maturation of the autophagic vacuole. J Cell Biol. 1990 Jun;110(6):1935–1945. [PMC free article] [PubMed]
  • Dunn WA., Jr Autophagy and related mechanisms of lysosome-mediated protein degradation. Trends Cell Biol. 1994 Apr;4(4):139–143. [PubMed]
  • Egner R, Thumm M, Straub M, Simeon A, Schüller HJ, Wolf DH. Tracing intracellular proteolytic pathways. Proteolysis of fatty acid synthase and other cytoplasmic proteins in the yeast Saccharomyces cerevisiae. J Biol Chem. 1993 Dec 25;268(36):27269–27276. [PubMed]
  • Furuno K, Ishikawa T, Akasaki K, Lee S, Nishimura Y, Tsuji H, Himeno M, Kato K. Immunocytochemical study of the surrounding envelope of autophagic vacuoles in cultured rat hepatocytes. Exp Cell Res. 1990 Aug;189(2):261–268. [PubMed]
  • Gordon PB, Høyvik H, Seglen PO. Prelysosomal and lysosomal connections between autophagy and endocytosis. Biochem J. 1992 Apr 15;283(Pt 2):361–369. [PubMed]
  • Güldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 1996 Jul 1;24(13):2519–2524. [PMC free article] [PubMed]
  • Harding TM, Hefner-Gravink A, Thumm M, Klionsky DJ. Genetic and phenotypic overlap between autophagy and the cytoplasm to vacuole protein targeting pathway. J Biol Chem. 1996 Jul 26;271(30):17621–17624. [PubMed]
  • Harding TM, Morano KA, Scott SV, Klionsky DJ. Isolation and characterization of yeast mutants in the cytoplasm to vacuole protein targeting pathway. J Cell Biol. 1995 Nov;131(3):591–602. [PMC free article] [PubMed]
  • Heinemeyer W, Gruhler A, Möhrle V, Mahé Y, Wolf DH. PRE2, highly homologous to the human major histocompatibility complex-linked RING10 gene, codes for a yeast proteasome subunit necessary for chrymotryptic activity and degradation of ubiquitinated proteins. J Biol Chem. 1993 Mar 5;268(7):5115–5120. [PubMed]
  • Holen I, Gordon PB, Seglen PO. Protein kinase-dependent effects of okadaic acid on hepatocytic autophagy and cytoskeletal integrity. Biochem J. 1992 Jun 15;284(Pt 3):633–636. [PubMed]
  • Holen I, Gordon PB, Seglen PO. Inhibition of hepatocytic autophagy by okadaic acid and other protein phosphatase inhibitors. Eur J Biochem. 1993 Jul 1;215(1):113–122. [PubMed]
  • Jones EW. Three proteolytic systems in the yeast saccharomyces cerevisiae. J Biol Chem. 1991 May 5;266(13):7963–7966. [PubMed]
  • Klionsky DJ, Cueva R, Yaver DS. Aminopeptidase I of Saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway. J Cell Biol. 1992 Oct;119(2):287–299. [PMC free article] [PubMed]
  • Knighton DR, Zheng JH, Ten Eyck LF, Ashford VA, Xuong NH, Taylor SS, Sowadski JM. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407–414. [PubMed]
  • Knighton DR, Zheng JH, Ten Eyck LF, Xuong NH, Taylor SS, Sowadski JM. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):414–420. [PubMed]
  • Kopitz J, Kisen GO, Gordon PB, Bohley P, Seglen PO. Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes. J Cell Biol. 1990 Sep;111(3):941–953. [PMC free article] [PubMed]
  • Kübrich M, Dietmeier K, Pfanner N. Genetic and biochemical dissection of the mitochondrial protein-import machinery. Curr Genet. 1995 Apr;27(5):393–403. [PubMed]
  • Lawrence BP, Brown WJ. Autophagic vacuoles rapidly fuse with pre-existing lysosomes in cultured hepatocytes. J Cell Sci. 1992 Jul;102(Pt 3):515–526. [PubMed]
  • Ogura K, Wicky C, Magnenat L, Tobler H, Mori I, Müller F, Ohshima Y. Caenorhabditis elegans unc-51 gene required for axonal elongation encodes a novel serine/threonine kinase. Genes Dev. 1994 Oct 15;8(20):2389–2400. [PubMed]
  • Punnonen EL, Autio S, Kaija H, Reunanen H. Autophagic vacuoles fuse with the prelysosomal compartment in cultured rat fibroblasts. Eur J Cell Biol. 1993 Jun;61(1):54–66. [PubMed]
  • Rabouille C, Strous GJ, Crapo JD, Geuze HJ, Slot JW. The differential degradation of two cytosolic proteins as a tool to monitor autophagy in hepatocytes by immunocytochemistry. J Cell Biol. 1993 Feb;120(4):897–908. [PMC free article] [PubMed]
  • Raths S, Rohrer J, Crausaz F, Riezman H. end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae. J Cell Biol. 1993 Jan;120(1):55–65. [PMC free article] [PubMed]
  • Riezman H. Endocytosis in yeast: several of the yeast secretory mutants are defective in endocytosis. Cell. 1985 Apr;40(4):1001–1009. [PubMed]
  • Riezman H. Yeast endocytosis. Trends Cell Biol. 1993 Aug;3(8):273–277. [PubMed]
  • Roberts CJ, Raymond CK, Yamashiro CT, Stevens TH. Methods for studying the yeast vacuole. Methods Enzymol. 1991;194:644–661. [PubMed]
  • Rose MD, Novick P, Thomas JH, Botstein D, Fink GR. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. [PubMed]
  • Schatz G, Dobberstein B. Common principles of protein translocation across membranes. Science. 1996 Mar 15;271(5255):1519–1526. [PubMed]
  • Schekman R. Genetic and biochemical analysis of vesicular traffic in yeast. Curr Opin Cell Biol. 1992 Aug;4(4):587–592. [PubMed]
  • Schekman R, Esmon B, Ferro-Novick S, Field C, Novick P. Yeast secretory mutants: isolation and characterization. Methods Enzymol. 1983;96:802–815. [PubMed]
  • Schekman R, Orci L. Coat proteins and vesicle budding. Science. 1996 Mar 15;271(5255):1526–1533. [PubMed]
  • Seglen PO, Bohley P. Autophagy and other vacuolar protein degradation mechanisms. Experientia. 1992 Feb 15;48(2):158–172. [PubMed]
  • Sengstag C. The sequence of Saccharomyces cerevisiae cloning vector pCS19 allowing direct selection for DNA inserts. Gene. 1993 Feb 14;124(1):141–142. [PubMed]
  • Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. [PubMed]
  • Stack JH, DeWald DB, Takegawa K, Emr SD. Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast. J Cell Biol. 1995 Apr;129(2):321–334. [PMC free article] [PubMed]
  • Stack JH, Horazdovsky B, Emr SD. Receptor-mediated protein sorting to the vacuole in yeast: roles for a protein kinase, a lipid kinase and GTP-binding proteins. Annu Rev Cell Dev Biol. 1995;11:1–33. [PubMed]
  • Takeshige K, Baba M, Tsuboi S, Noda T, Ohsumi Y. Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. J Cell Biol. 1992 Oct;119(2):301–311. [PMC free article] [PubMed]
  • Tan JL, Spudich JA. Characterization and bacterial expression of the Dictyostelium myosin light chain kinase cDNA. Identification of an autoinhibitory domain. J Biol Chem. 1991 Aug 25;266(24):16044–16049. [PubMed]
  • Teichert U, Mechler B, Müller H, Wolf DH. Lysosomal (vacuolar) proteinases of yeast are essential catalysts for protein degradation, differentiation, and cell survival. J Biol Chem. 1989 Sep 25;264(27):16037–16045. [PubMed]
  • Thumm M, Egner R, Koch B, Schlumpberger M, Straub M, Veenhuis M, Wolf DH. Isolation of autophagocytosis mutants of Saccharomyces cerevisiae. FEBS Lett. 1994 Aug 1;349(2):275–280. [PubMed]
  • Tooze J, Hollinshead M, Ludwig T, Howell K, Hoflack B, Kern H. In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome. J Cell Biol. 1990 Aug;111(2):329–345. [PMC free article] [PubMed]
  • Tsukada M, Ohsumi Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett. 1993 Oct 25;333(1-2):169–174. [PubMed]
  • Tuttle DL, Dunn WA., Jr Divergent modes of autophagy in the methylotrophic yeast Pichia pastoris. J Cell Sci. 1995 Jan;108(Pt 1):25–35. [PubMed]
  • Ueno T, Muno D, Kominami E. Membrane markers of endoplasmic reticulum preserved in autophagic vacuolar membranes isolated from leupeptin-administered rat liver. J Biol Chem. 1991 Oct 5;266(28):18995–18999. [PubMed]
  • Van Den Hazel HB, Kielland-Brandt MC, Winther JR. Review: biosynthesis and function of yeast vacuolar proteases. Yeast. 1996 Jan;12(1):1–16. [PubMed]
  • Wach A, Brachat A, Pöhlmann R, Philippsen P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast. 1994 Dec;10(13):1793–1808. [PubMed]
  • Woolford CA, Daniels LB, Park FJ, Jones EW, Van Arsdell JN, Innis MA. The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases. Mol Cell Biol. 1986 Jul;6(7):2500–2510. [PMC free article] [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)