Stt4 directly interacts with Ypp1 to form a stable complex. (A) Fluorescent image of GFP-Stt4 (left), Ypp1-GFP (right), and their corresponding kymographs (bottom). A representative section of the cell (the demarcated region in top panels) was examined for its relative movement at the cell membrane. A fluorescent time point of the demarcated region was taken every 5 s over the course of 3 min. The spatial variance of the selected fluorescent region with regard to time is represented in the bottom image. (B) FRAP of the PtdIns4P reporter GFP-2xPH^Osh2 (top and Video 3, available) and GFP-Stt4 (bottom and Video 2). The white partitions demarcate the regions exposed to high intensity light, a FRAP region, and a control region exposed to normal excitation. The corresponding graph quantifies the fluorescent values with regard to time. (C) Cells expressing HA-Ypp1 and GFP alone or GFP-Stt4 were grown to mid-log phase, lysed, and incubated with an anti-GFP antibody. Coimmunoprecipitated proteins were detected with an anti-HA antibody. The relative abundance of coimmunoprecipitated HA-Ypp1 is shown in the top panel compared with a sample of the input from the whole cell lysate (WCL) in the bottom panel. Note that a small region of the immunoprecipitated GFP-Stt4 is cleaved at the C terminus. (D) Truncation mutants of GST-tagged Stt4 were purified from E. coli lysates, immobilized on glutathione beads, and normalized to equal amounts. Beads containing the GST-Stt4 truncations were then incubated for 1 h with lysate from E. coli overexpressing HIS₆-Ypp1. Protein that bound the GST-Stt4 fragments was probed using an anti-HIS₆ antibody. The relative amount of HIS₆-Ypp1 that bound each respective GST-Stt4 fragment (top) and the relative amount of the Stt4 fragments that bound the glutathione beads (bottom) are shown. Notice that the primary Stt4 fragment (736–1346) is a C-terminal degradation product missing ∼200 amino acids. C-terminal degradation products are marked with an asterisk.