Characterization of a novel autophagy-specific gene, ATG29

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Abstract

Autophagy is a process whereby cytoplasmic proteins and organelles are sequestered for bulk degradation in the vacuole/lysosome. At present, 16 ATG genes have been found that are essential for autophagosome formation in the yeast Saccharomyces cerevisiae. Most of these genes are also involved in the cytoplasm to vacuole transport pathway, which shares machinery with autophagy. Most Atg proteins are colocalized at the pre-autophagosomal structure (PAS), from which the autophagosome is thought to originate, but the precise mechanism of autophagy remains poorly understood. During a genetic screen aimed to obtain novel gene(s) required for autophagy, we identified a novel ORF, ATG29/YPL166w. atg29Δ cells were sensitive to starvation and induction of autophagy was severely retarded. However, the Cvt pathway operated normally. Therefore, ATG29 is an ATG gene specifically required for autophagy. Additionally, an Atg29-GFP fusion protein was observed to localize to the PAS. From these results, we propose that Atg29 functions in autophagosome formation at the PAS in collaboration with other Atg proteins.

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Materials and methods

Yeast strains and media. Growth media, culture conditions, and genetic manipulations were all as previously described [9]. The yeast strains used in this study are listed in Table 1. A deletion construct to disrupt ATG29 with kanMX was amplified by PCR from BY4741-based atg29 deletion mutants [10]. The resultant cassette was transformed into SEY6210, BJ2169, and KVY55 strains to make TMK4, TMK368, or TMK17, respectively. TMK372 was made by replacing ATG8 gene with the LEU2 gene [11]. TMK105 was

Identification of ATG29/YPL166w

All previously discovered atg mutants, which are defective in autophagy, show a loss of viability under starvation conditions. Thus, sensitivity to starvation would seem to be a good indicator of autophagy defects [15]. To search for novel genes involved in autophagy, we performed a genome-wide screen using a collection of roughly 4500 yeast single-gene deletion mutants in the BY4741 background [10]. Each strain was first screened for the reduced viability under starvation conditions and over

Discussion

The yeast, S. cerevisiae, represents an excellent system for studying the molecular mechanisms of autophagy. In this organism, the machinery used in autophagy is similar to that used by the Cvt pathway. Among the 27 ATG genes involved in the Cvt pathway and/or autophagy, ATG17 is the only gene known to function specifically in autophagy. Recently, novel ATG genes required only for the Cvt pathway were isolated by screening for defects in proAPI maturation [12], [22]. In contrast, it is more

Acknowledgments

We thank the members of the Ohsumi laboratory for helpful discussion, and the National Institute for Basic Biology Center for Analytical Instruments for technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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    Abbreviations: PAS, pre-autophagosomal structure; ATG, autophagy-related genes; Cvt, cytoplasm to vacuole targeting; API, aminopeptidase I; GFP, green fluorescent protein; Vps, vacuolar protein-sorting; PE, phosphatidylethanolamine.

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