Figure 2. 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 mem- brane. 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-2xPHOsh2 (top and Video 3, available at http://www .jcb.org/cgi/content/full/jcb.200804003/DC1) 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. Coimmuno- precipitated 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. doxycycline, an epitope-tagged Ypp1-HA is no longer detected in cell extracts (Fig. 3 B). Despite low amounts of the essential Ypp1 protein, teto-YPP1 cells remain viable and recover once doxycycline is removed. We used teto-YPP1 and control strains to measure the rela- tive abundance of the various phosphoinositides by incorporating 3H-labeled myo-inositol into live cells, isolating and deacylat- ing the lipids, and separating the different deacylated inositol species with HPLC. Doxycycline-treated teto-YPP1 cells have PtdIns4P levels reduced to half the relative amount measured in vehicle-treated teto-YPP1 cells (Fig. 3 C). This effect is specific to Ypp1 depletion as treatment of the control strain, R1158, with doxycycline showed no decrease of PtdIns4P levels. Consistent with earlier findings regarding inactivation of stt4, the down- stream lipid PtdIns(4,5)P2 levels also decrease (Audhya et al., 2000). Similar lipid profiles were observed in experiments using the various ypp1ts strains at restrictive temperatures (un- published data). Ypp1 selectively impacts the PM pool of PtdIns4P Our focus turned toward studying steady-state Stt4 activity in the absence of Ypp1. For this, we isolated a bypass suppressor of ypp1 , the PtdIns 4-phosphatase SAC1. We found that yeast lacking SAC could bypass deletion of YPP. Additionally, sac1 could bypass stt4 . The relative growth strength of these vari- ous strains demonstrate that the sac1 ypp1 yeast are slightly healthier than sac1 stt4 , implying that the PtdIns 4-kinase may retain some function in the absence of Ypp1 (Fig. 4 A). To further analyze the importance of this finding, we measured PIP levels of each strain. Deletion of SAC1, the primary PtdIns 4-phosphatase for PM-generated PtdIns4P (Guo et al., 1999; Foti et al., 2001), results in a dramatic increase in PtdIns4P levels, tenfold over wild type (Fig. 4 B). However, the double deletion sac1 ypp1 reduces that elevation to roughly twofold wild type. PtdIns4P is further reduced in the sac1 stt4 strain so that it 1064 JCB • VOLUME 183 • NUMBER 6 • 2008