Figure 7.

Rcy1 interacts with Snc1 and together with Ypt31/32 affects the level of Snc1 phosphorylation. (A) Ypt31/32 and Rcy1 affect the phosphorylation state of Snc1. Wild-type and mutant cells expressing GFP-Snc1 were grown at 26°C. Cell lysates were prepared and assayed by Western blot analysis by using anti-Snc1 antibodies. The higher molecular weight band is the phosphorylated form of Snc1, whereas the lower molecular weight band is the unphosphorylated form (Galan et al., 2001). The bands were quantified using an Alpha Imager, and the percentage of phosphorylated Snc1 averaged from three independent experiments is indicated; ± represents the SEM. (B) Rcy1 interacts with Snc1. Cell lysates were prepared from wild-type cells expressing GST (NSY784) or GST-Rcy1 (NSY624) as described in Materials and Methods. Cell lysates (two left lanes, 25% of the cell lysate used for pull-down) and GST-pull down fraction (two right lanes) were assayed by Western blot analysis. GST proteins were detected using a polyclonal anti-GST antibody, and Snc1 protein was detected using a polyclonal anti-Snc1 antibody. The results shown are representative of two experiments. Snc1 occurs as two bands in A and one band in B because the separation of the two bands requires electrophoresis in a high percent gel (15%) for a long period (overnight at 4°C), which was not done in B.