Tor-dependent phosphorylation of Apg13 inhibits Apg1–Apg13 association. A, Bandshift of Apg13 in response to starvation and rapamycin. YEPD-grown cells (BJ2168) expressing APG13 with YEp352[APG13] (lanes 1 and 6) were transferred to SD(−N) medium (lanes 2–5) or treated with 0.2 μg/ml rapamycin (lanes 7–10) and incubated for the indicated times. Total protein was analyzed by immunoblot using anti-Apg13 serum. B, Apg13 is hyperphosphorylated. Cells (TFD13-W3) overexpressing APG13 were labeled in vivo with 35S (lanes 1–6) or 32Pi (lanes 7 and 8), and shifted to YEPD (lane 5) or SD(−N) (lanes 6 and 8) for 1 h. Apg13 protein was immunoprecipitated and treated with alkaline phosphatase (PPase; lane 4). Immunoprecipitated protein was subjected to SDS-PAGE, followed by autoradiography. Cont, immunoprecipitation was carried out with preimmune serum. C, Tor-mediated phosphorylation/dephosphorylation of Apg13. Wild-type (WT, JK9-3da) or TOR1-1 (JH11-1c) cells expressing Apg13 grown in YEPD were treated with or without 0.2 μg/ml of rapamycin for 1 h. An immunoblot using anti-Apg13 serum was carried out. D, Phosphorylation/dephosphorylation cycle of Apg13 in response to nutrient conditions. YEPD-grown cells (BJ2168) overexpressing Apg13 (lane 1) were incubated in SD(−N) for 1.5 h (lane 2) with or without 30-min treatment with 10 μg/ml cycloheximide (lanes 4 and 5), 0.2 μg/ml of rapamycin (lanes 6 and 7), or both (lanes 8 and 9). Then, half a sample volume of 2× YEPD was added to the cells, after which they were incubated for 10 min (lanes 3, 5, 7, and 9). Apg13 protein was detected by immunoblot. E, Coimmunoprecipitation of Apg13 with Apg1. YEPD grown cells (BJ2168) overexpressing HAApg1 and/or Apg13 with high-copy plasmids pRS426[HA APG1] (p[HAAPG1]) and YEp351[APG13] (p[APG13]) were lysed, and HAApg1 was immunoprecipitated (IP) from the cell lysate. Apg13 and HAApg1 were detected by immunoblot (lanes 1–6). Apg13 and HAApg1 detected in total cell lysate are also shown (lanes 7–9). The hyperphosphorylated form of Apg13 remaining in the supernatant of the immunoprecipitate was still detected (data not shown), excluding the possibility that Apg1-bound Apg13 was dephosphorylated during the experiment. The asterisk shows a band that anti-HA ascite nonspecifically recognized in total lysate. F, Apg1–Apg13 association is promoted by rapamycin treatment. YEPD-grown cells (BJ2168) expressing HAApg1 and Apg13 with low-copy plasmids pRS316[HA APG1] and pRS315[APG13] were enzymatically converted to spheroplasts and treated with 0.2 μg/ml of rapamycin (at time 0). Spheroplasts were harvested at the indicated times, and coimmunoprecipitation was performed. Apg1 protein kinase assay of the immunoprecipitates was also performed (bottom).