Figure 1.
Atg29 interaction with Atg17 is essential for autophagy. (A) The Atg17 binding site in Atg29. The indicated segments of Atg29 were fused to the Gal4 DNA-binding domain (BD). Binding to Atg17 fused to the Gal4-activating domain was evaluated by growth on −Ade plates for 3 d. (B) The Atg29 binding sites in Atg17. The indicated segments of Atg17 were fused to the Gal4 (transcriptional) activation domain (AD). Binding to Atg29 was tested by growth on −His plates containing 3 mM 3-AT. Atg17 interacts with Atg29 via coiled-coil domain 2. (C) Mutant Atg29s (Atg29W54R, Atg29P64H, Atg29F67L, or Atg29R71G) did not associate with Atg17. Two-hybrid assays were carried out as described in A. (D) Mutant Atg29s failed to interact with Atg17 in vivo. atg29Δ (TMK4) cells carrying the indicated myc-tagged ATG29 plasmids (wild-type and mutants) were grown in YEPD, and then they were treated with 0.2 μg/ml rapamycin for 3 h. Cell extracts were immunoprecipitated with anti-myc antibody. Coimmunoprecipitated proteins were analyzed by immunoblotting with anti-myc (top) or anti-Atg17 antibodies (middle). The bottom row indicates the amount of Atg17 in the total lysates. (E) The binding of Atg29 and Atg17 is essential for autophagy. Wild-type (BY4741 + pTN3) or atg29Δ (TMK181 + pTN3) cells carrying pTM108 (Atg29), pTM109 (Atg29F67L), pTM110 (Atg29R71G), or pRS313 (empty vector) were cultured in SCD, and then they were transferred to SD(−N) for 4 h, lysed, and assayed for ALP activity. The bars represent the SD of three independent experiments. (F) Atg17 is required for proper Atg29 localization to the PAS. WT (TMK190) or atg17Δ (TMK214) cells were grown in SD medium containing casamino acid supplemented with adenine, tryptophan, and uracil, and then they were treated with 0.2 μg/ml rapamycin. Growing (0 h) and rapamycin-treated (3 h) cells were observed by fluorescence microscopy. The percentages of cells with fluorescent dot signals at the PAS were determined. Bar, 5 μm. (G) Atg29 is not required for proper Atg17 localization to the PAS. WT (TMK576) or atg29Δ (TMK600) cells were tested as in F.