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. 2009 Dec 7;30(4):1049–1058. doi: 10.1128/MCB.01344-09

FIG. 2.

FIG. 2.

TORC1 directly phosphorylates Atg13 in vitro. (A) Detection of immunoprecipitated TORC1. FlagKog1 was immunoprecipitated, and wild-type HATor1 (WT) and a kinase-dead D2294E mutant (KD) contained in the immunocomplex were detected. Results of a control experiment using untagged Kog1 are shown in lane 1. α, anti. (B) In vitro TORC1 kinase assay. TORC1 isolated as for panel A was used in an in vitro kinase assay using 4 μg of recombinant trigger factor (TF)-fused Atg13 (wild type [lanes 1 to 3] or Atg13-8SA [lanes 4 to 6]) or TF (lane 7) as substrates. The results of autoradiography (kinase assay) and Coomassie blue staining (CBB) are shown. (C) TORC1 kinase assay using various Atg13 constructs. TORC1 assay was performed using the indicated TF-Atg13 proteins. TF-Atg13-4SAb and TF-Atg13-7SAb migrated faster than the wild type, presumably because they were C-terminally processed in E. coli cells.