Biochemical and Biophysical Research Communications
Characterization of the Atg17–Atg29–Atg31 complex specifically required for starvation-induced autophagy in Saccharomyces cerevisiae☆
Section snippets
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.
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Present address: Division of Evolutionary Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji-cho, Okazaki 444-8585, Japan.