Figure 4. Deletion of SAC1 bypasses ypp1 or stt4 . (A) Relative growth proﬁles of wild type, sac1 , sac1 stt4 , sac1 ypp1 , stt4 , and ypp1 at 26°C. (B) Phosphoinositide levels of wild type, sac1 , sac1 stt4 , and sac1 ypp1 . Data are presented as means and SD (error bars) of three indepen- dent experiments. (C) Cellular localization of the PtdIns4P reporter GFP-2xPHOsh2 in wild type, pik1ts, and stt4ts (top) at the restrictive temperature. (bottom) The ﬂuorescent localization of GFP-2xPHOsh2 in teto-YPP1 grown in the presence (bottom) or absence (top) of doxycycline for 20 h. Bars, 4 μm. delocalized from PM PIK patches to the diffuse cytoplasm (Fig. 5 B). Similarly, in teto-STT4 GFP-YPP1 (Fig. 5 B), the ad- dition of doxycycline resulted in redistribution of GFP-Ypp1 to the cytoplasm. These results were also observed using sac1 stt4 GFP-YPP1 (unpublished data). We conclude that Ypp1 and Stt4 rely on each other to maintain their localization such that recruitment and stability of the PIK patch at the PM de- pends on an intact Stt4–Ypp1 complex. Can Ypp1 organize into PIK patches in the absence of Stt4? This question was addressed by artificially targeting Ypp1 to the PM in the absence of Stt4 and studying PIK patch formation. We fused a palmitoylation signal (the first 28 amino acids of Psr1) to the N terminus of full-length GFP-Ypp1 (Siniossoglou et al., 2000). Although this construct failed to rescue ypp1 cells, it localized similarly to Ypp1 in a wild- type background (Fig. 5 C, top left) and colocalized with mCherry-Stt4 (Fig. S3, available at http://www.jcb.org/cgi/ content/full/jcb.200804003/DC1). The absence of Stt4, how- ever, in a sac1 stt4 PSR11-28-GFP-YPP1 released Ypp1 from the wild-type cortical PIK patches to a uniform PM distribu- tion. The inverse experiment using sac1 ypp1 PSR11-28- GFP-STT4 failed to generate any significant fluorescent signal or maintain any significant protein levels as determined by Western blot. We conclude that Ypp1 is necessary to structur- ally stabilize Stt4, which in turn allows the complex to orga- nize and be recruited to the PM. An Stt4–Ypp1 complex forms the core of the PIK patch We used differential centrifugation to fractionate the various subcellular components of yeast to compare relative PIK patch densities with known cellular components. HA-Ypp1 fraction- ates into the membrane-associated P100 fraction in a wild-type background (Fig. 6 A). However, deletion of STT4 in the sac1 background completely redistributes HA-Ypp1 from P100 frac- tion to the S100 fraction, indicating that its normal distribution to membrane-bound structures depends on Stt4 (Fig. 6 A, right). We collected this soluble fraction and determined the relative size of a Ypp1 protein complex by measuring its elution from a Super- dex S300 column in the absence of Stt4. Soluble HA-Ypp1 (99 kD) from the stt4 sac1 strain was stable and eluted in several fractions with a peak corresponding to a molecular mass of 150 kD (Fig. 6 B). The fast protein liquid chromatography (FPLC) elution profile suggests that Ypp1 may dimerize or bind to other PIK patch-associated components. We tested Ypp1’s capability to dimerize/oligomerize in yeast extracts. Coimmunoprecipitation of HA-Ypp1 with GFP alone, GFP-Ypp1, or GFP-Stt4 demonstrates that HA-Ypp1 interacts well with both GFP-Stt4 and GFP-Ypp1 compared with GFP alone (Fig. 6 C). To test if the interaction between HA- Ypp1 and GFP-Ypp1 is direct or indirect through Stt4, we re- peated the experiment in the sac1 stt4 background. In stt4 sac1 yeast lysates, HA-Ypp1 no longer coimmunoprecipitates 1066 JCB • VOLUME 183 • NUMBER 6 • 2008