Figure 10.

In vitro phosphorylation of Gcn4 by Pho85-Pcl1. Different substrates (indicated at the bottom) were incubated with wild-type or catalytically inactive (E53A) GST-Pho85, with or without GST-Pcl1. Asterisk indicates background phosphorylation on bacterial proteins copurifying with the recombinant kinase and cyclin. The two arrowheads indicate the two phosphorylation bands of GST-Gcn4^62–202. The two weak signals (<10% intensity) visible over the main band in the T165A mutant presumably represent phosphorylation at alternative sites. The right panel shows that GST alone is not phosphorylated by Pho85-Pcl1 (the GST protein band would be expected to migrate slightly faster than the 30-kDa marker).