Characterization of the Atg17–Atg29–Atg31 complex specifically required for starvation-induced autophagy in Saccharomyces cerevisiae

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Abstract

Nutrient starvation induces autophagy to degrade cytoplasmic materials in the vacuole/lysosomes. In the yeast, Saccharomyces cerevisiae, Atg17, Atg29, and Atg31/Cis1 are specifically required for autophagosome formation by acting as a scaffold complex essential for pre-autophagosomal structure (PAS) organization. Here, we show that these proteins constitutively form an Atg17–Atg29–Atg31 ternary complex, in which phosphorylated Atg31 is included. Reconstitution analysis of the ternary complex in E. coli indicates that the three proteins are included in equimolar amounts in the complex. The molecular mass of a monomeric Atg17–Atg29–Atg31 complex is calculated at 97 kDa; however, analytical ultracentrifugation shows that the molecular mass of the ternary complex is 198 kDa, suggesting a dimeric complex. We propose that this ternary complex acts as a functional unit for autophagosome formation.

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Materials and methods

Yeast strains and media. BY4741 (MATahis3Δleu2Δmet15Δura3Δ) (ResGen), YKY61 (BY4741 atg31::ATG31–13Myc-kanMX) [6], YKY69 (YKY61 atg17Δ::HIS3), YKY71 (YKY61 atg29Δ::natMX), and YKY88 (BY4741 atg17Δ::kanMX atg31Δ::natMX) were used in this study. The other atg disruptants listed in Fig. 2B were purchased from ResGen. Media and methods for gene disruption have been described previously [7], [8].

Plasmids. To obtain pATG31, a 1.6-kb fragment containing the entire ATG31 gene was cloned from yeast

Atg17, Atg29, and Atg31/Cis1 form a stable complex in the cytosol

Recently, Atg29 and Atg31/Cis1 have been individually reported to interact with Atg17 [6], [11]. We thus examined the behavior of Atg17, Atg29, and Atg31 in vivo. Fractionation analysis of wild-type cells under growth conditions showed that the majority of Atg17, Atg29, and Atg31 were present in the cytosolic fraction (data not shown). This fraction was subjected to gel filtration analysis using a Superdex 200 column. Atg17 eluted mainly in a single peak in fractions corresponding to ∼600 kDa (

Acknowledgments

We thank H. Nakatogawa (in our laboratory) for helpful discussion and the National Institute for Basic Biology Center for Analytical Instruments for technical assistance.

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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.

1

Present address: Division of Evolutionary Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji-cho, Okazaki 444-8585, Japan.

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