Elsevier

Gene

Volume 192, Issue 2, 19 June 1997, Pages 245-250
Gene

Apg1p, a novel protein kinase required for the autophagic process in Saccharomyces cerevisiae

https://doi.org/10.1016/S0378-1119(97)00084-XGet rights and content

Abstract

Autophagic protein degradation includes bulk protein turnover with dynamic membrane reorganization, in which formation of novel organelles autophagosomes play key roles. We have shown that Saccharomyces cerevisiae performs the autophagy in the vacuole, a lytic compartment of yeast, in response to various kinds of nutrient starvation. Here we show that the APG1 gene, involved in the autophagic process in yeast, encodes a novel type of Ser/Thr protein kinase. Our results provide direct evidence for involvement of protein phosphorylation in regulation of the autophagic process. We found overall homology of Apg1p with C. elegans Unc-51 protein, suggesting that homologous molecular mechanisms, conserved from unicellular to multicellular organisms, are involved in dynamic membrane flow.

Introduction

Vacuolar/lysosomal protein degradation plays crucial roles in turnover of cellular components. Proteins that are destined for degradation are transported by several mechanisms, and autophagy has been shown to be the major route of delivery of cytoplasmic proteins into vacuoles/lysosomes under conditions where cells require enhanced protein degradation and remodeling of components (Mortimore, 1987; Dunn, 1994). The autophagic pathway includes formation of autophagosomes and maturation of autophagosomes to autolysosomes, following degradation within autolysosomes. We have little knowledge, however, about machinery and regulation of the autophagy, because the autophagic process, which comprises complicated steps and proceeds rapidly, are not suited for biochemical analysis in mammalian systems.

Recent studies have shown that the yeast S. cerevisiae is useful model organism for studying protein degradation. Besides well-characterized non-lysosomal protein degradation mediated by the ubiquitin-proteasome system (Jentsch, 1992), this organism possesses a vacuole, an acidic compartment which is functionally equivalent to animal lysosome (for review, see Klionsky et al., 1990). We have reported that cytosolic components of yeast cells are degraded in the vacuole through the autophagic process, and that this process is equivalent to macroautophagy in mammalian cells (Takeshige et al., 1992; Baba et al., 1994).

As autophagy of yeast is induced by depletion of nutrients such as nitrogen, carbon, sulfur, or amino acids, we can obtain yeast cells carrying out the autophagy uniformly and on a large scale. Furthermore, progression of yeast autophagy is easily monitored under a light microscope. Upon a condition where vacuolar protease activity is repressed by the addition of a protease inhibitor PMSF, or by genetic manipulations to be vacuolar protease activity-deficient, accumulation of autophagic bodies, the final intermediate structures sequestering cytosolic components to the vacuoles, are observed under a phase-contrast microscope (Takeshige et al., 1992). Taking advantage of these features, we have taken a genetic approach to isolate apg mutants that have a defect in the autophagic process (Tsukada and Ohsumi, 1993). Here we report structural analyses of the APG1 gene. The APG1 gene product, Apg1p, is shown to encode a protein kinase. Our results provide direct evidence that the autophagy in yeast is regulated by protein phosphorylation.

Section snippets

Results and discussion

The wild-type APG1 gene was cloned by complementation of Apg phenotype of an apg1-1 mutant. Integrative mapping analysis demonstrated that the cloned DNA contained a region corresponding to the apg1 mutation locus. Nucleotide sequence analysis of the 3.6-kb XbaI-BamHI fragment sufficient to complement the apg1 mutation identified a single open reading frame (ORF) of 2691 bp (Fig. 1a). This ORF, found to be identical to YGL180w, has a potential to encode a protein of 897 amino acid residues

Acknowledgements

We thank A. Toh-e for providing plasmids and anti-myc monoclonal antibody, and M. Fujino for advice on protein kinase assay. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan and by Special Condition Funds for the Promotion of Science and Technology from the Science and Technology Agency of Japan. A.M. was supported by the JSPS Fellowships for Young Scientists.

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    Present address: Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226, Japan.

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