Figure 2.

Genetic and biochemical interactions between Atg1 and spaghetti-squash activator (Sqa). (A–K) Genetic interactions between Atg1, Sqa, and spaghetti-squash (Sqh). Compared with the ptc-GAL4 controls (A), expression of Atg1 or Sqa by ptc-GAL4 resulted in missing anterior cross-vein phenotypes (B, C). However, RNAi-mediated downregulation of Atg1 (Atg1^RNAi) or Sqa (Sqa^RNAi) did not cause wing vein defects (D, E). Depletion of Atg1 and Sqa suppressed Atg1 and Sqa-induced wing vein defects, respectively (F, I). Atg1-induced wing defects were modulated by depletion of Sqa (G) or by co-expression of Sqh^A20A21 (H). Nevertheless, Sqa-induced wing vein defects were suppressed by co-expression of Sqh^A20A21 (J) but not Atg1^RNAi (K). (L) Schematic presentation of the domain structures of Sqa and deletion mutants. (M–N) Atg1 physically interacted with Sqa. (M) 293T cells were transfected with HA-tagged Sqa WT (wild-type) or KA, together with Flag-tagged Atg1 WT or KR. The cells were lysed 48 h after transfection and immunoprecipitated (IP) with anti-Flag antibodies. The immunoprecipitated proteins and the total cell lysates (TCL) were analysed by immunoblotting (IB) with antibodies as indicated. (N) 293T cells transfected with Flag–Atg1-KR and various HA-tagged Sqa contracts were subjected to immunoprecipitations with anti-HA antibody. The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated. (O) Atg1 directly phosphorylated Sqa in vitro. Flag-tagged Atg1 WT or KR immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant Sqa-K1, Sqa-K2, and Sqa-C in an in vitro kinase assay. The lower panels represent equal input of His-fusion proteins and Atg1 immunoprecipitates.