Figure 7. Efr3 is a component of PIK patches at the PM. (A) Tetrad dissection of diploid yeast with a deleted chromo- somal copy of efr3. (B) Full-length and truncation mutants of GST-tagged Efr3 were puriﬁed from E. coli lysates, immobi- lized on glutathione beads, and normalized to equal amounts. Beads containing the GST-Efr3 constructs were then incubated for 1 h with yeast lysate expressing Ypp1-GFP. Protein that bound the GST-Efr3 fragments was probed using an anti-GFP antibody. (C) Subcellular fractionation of Efr3-GFP from wild- type cell extracts. (D) Cells coexpressing Efr3-GFP and Ypp1- mCherry were grown at 26°C and examined by ﬂuorescence microscopy. Efr3-GFP localizes to cortical patches on the PM (left) and with a similar distribution as Ypp1-mCherry (right). Arrows on the Efr3-GFP image indicate regions of colocalization. Bar, 4 μm. Second, our analysis of temperature-sensitive mutant alleles of stt4 and ypp1 demonstrated a synthetic lethal interaction, indicating that the two protein products were involved in the same or parallel pathways. Third, using in vitro binding experi- ments with E. coli–expressed proteins, we established that Ypp1 directly binds multiple regions of Stt4. Finally, by placing YPP1 under the control of a doxycycline-repressible promoter, we showed that in the absence of Ypp1, Stt4 mislocalized to the diffuse cytoplasm and was subsequently degraded causing PtdIns4P levels to drop dramatically at the PM. Similarly, we found that attenuating Stt4 expression caused Ypp1 to delocalize from the PM. However, the Ypp1 protein remained intact and was not degraded. Therefore, we suggest that an intact Stt4– Ypp1 complex is necessary for PM recruitment and subsequent organization into a PIK patch. Our characterization of Ypp1 and Stt4 indicated that addi- tional factors might control PIK patch organization. During the course of this work, a genome-wide study suggested Efr3 as a candidate Ypp1-interacting protein (Tarassov et al., 2008). We have confirmed Efr3 as a PIK patch component and charac- terized its role in Stt4 PIK patch assembly. A previous study identified a synthetic lethal genetic interaction between PHO85 and EFR3 (PHO eighty five requiring; Lenburg and O’Shea, 2001). Interestingly, Pho85 is a member of the cyclin-dependent kinase family and mammalian Cdk5 has been implicated in PIP metabolism (Lee et al., 2004, 2005). We found that Efr3 is es- sential, similar to Ypp1 and Stt4. We showed that Efr3 interacts with Ypp1 and colocalizes with Stt4 and Ypp1 at PIK patches on the PM. Additionally, we generated an efr3ts allele and dem- onstrated that at nonpermissive temperatures PtdIns4P levels dropped dramatically at the PM. Finally, we showed that upon depletion of the Efr3 protein, GFP-Stt4 mislocalized from the PM. Importantly, depletion of Ypp1 (or Stt4) did not mislocal- ize Efr3-GFP from the PM but instead allowed it to adopt a more uniform distribution around the membrane. We propose that Efr3 acts as a PM anchor for Stt4 PIK patches and the Stt4– Ypp1 oligomeric complex stabilizes the distinctive clustering of Stt4 in the PIK patch. YPP1 AND EFR3 MEDIATE PIK PATCH ORGANIZATION • Baird et al. 1069