Fig. 4.

GTA activates the Atg1 kinase in an Atg11-dependent manner. (A) FLAG-Atg1 was immunoprecipitated from atg18Δ atg19Δ (referred to as WT) or atg11Δ atg18Δ atg19Δ cells with or without 0.04% MMS. Kinase activity was determined by incubation with MBP and ATP-γ³²P. To improve the efficiency of Atg1 complex immunoprecipitation from cell lysates, ATG18 was deleted to block autophagosome degradation. Because a high proportion of Atg1 kinase activity in cells arises from Atg19-bound cargo, ATG19 was also deleted to accurately determine GTA-specific activation of Atg1. Note that ATG19 is dispensable for GTA (Fig. 5B). (B) GTA partially requires Atg11’s receptor binding domain. WT, atg11Δ, and cells expressing Atg11 lacking the receptor-binding domain (atg11ΔRBD) were transformed with GFP-Atg8 and analyzed for GTA. A representative blot and quantification are shown. Error bars reflect SEM from three independent experiments.