Fig. 6. Phosphorylation of Sso in vitro by Tpk1 inhibits SNARE assembly. (A) Sso11–265 is phosphorylated in vitro by Tpk1. Purified GST–Sso1 was incubated with or without recombinant Tpk1 in the presence of [γ-32P]ATP. Samples were resolved by SDS–PAGE and detected by Coomassie Blue staining or autoradiography. (B) Phosphorylated Sso11–265 is deficient in its ability to form complexes with Sec9402–651. In vitro phosphorylated and non-phosphorylated GST–Sso11–265 were incubated with GST–Sec9402–651, after which samples were immunoprecipitated, resolved by SDS–PAGE and quantified by antibody detection. (C) SIT4 overexpression partially rescues snc cells. snc null cells transformed with a plasmid expressing Sit4 (+SIT4), or a control plasmid (snc), were grown and plated onto SC (35°C) and rich medium (YPD) with (+C2) or without (–C2) added C2-ceramide. Cells were grown for 36 h.