Figure 5. Stt4 and Ypp1 are codependent for proper localization and organization into PIK patches at the PM. (A) Relative abundance of GFP-Stt4 after doxycycline treatment in teto-YPP1 cells over the time course of 16 h. The relative abundance of G6PDH is shown as a control. (B) The localization proﬁle of teto-YPP1 GFP-Stt4 in the absence (top) or presence (second panel) of doxycycline and the corresponding DIC image. The localization teto-STT4 GFP-Ypp1 in the absence (third panel) or presence (bottom) of doxycycline and the corresponding DIC images. Bars, 4 μm. (C) Localization of PM anchored Psr11-28- GFP-Ypp1 expressed under wild-type conditions (left) or in the sac1 stt4 background (right). The graph is the relative ﬂuorescence intensity along the PM within the indicated spatial region. Arrows indicating the start and end of the ﬂuorescent images correspond to the x axis of the graph. with GFP-Ypp1 compared with lysates in which only Sac1 is deleted (Fig. 6 D). Ypp1’s inability to directly interact with it- self was confirmed with E. coli–purified HIS6-Ypp1. In the absence of any endogenous binding partners, the FPLC elution profile of purified HIS6-Ypp1 was consistent with a monomer (Fig. 6 E). Therefore, the Ypp1 oligomer requires Stt4. Addi- tionally, the high molecular mass elution profile of HA-Ypp1 isolated from yeast lysates (Fig. 6 B) indicates that HA-Ypp1 has other soluble binding partners in the absence of Stt4. The rolling blackout (RBO) yeast homologue Efr3 recruits the PIK patch to the PM Our results indicated that Ypp1 played an essential role in stabi- lizing Stt4 in the PM PIK patch. However, because neither Stt4 nor Ypp1 contain a recognizable membrane-targeting motif, we predicted that there were additional components critical for PIK patch membrane association. Accordingly, during the course of our Ypp1 characterization, a genome-wide protein interaction study uncovered Efr3 as another candidate component of the Stt4 PIK patch (Tarassov et al., 2008). Similar to STT4 and YPP1, we found that EFR3 is an essential gene (Fig. 7 A). Primary sequence analysis of Efr3 revealed potential homo- logues in higher eukaryotes, including humans. The best char- acterized of these is the Drosophila melanogaster RBO gene product, a multipass transmembrane protein essential for viabil- ity (Faulkner et al., 1998). Recent work has shown the RBO protein to be localized to presynaptic boutons of Drosophila neuromuscular junction synapses, where it has been suggested to have an essential role in synaptic vesicle exocytosis (Huang et al., 2004, 2006). Multiple lines of evidence indicate that Efr3 is a compo- nent of the Stt4 PIK patch. First, Ypp1 and Efr3 interact in vitro based on the ability of E. coli–purified GST-Efr3 fusions to bind Ypp1-GFP from yeast lysates. Full-length Efr3 and a C-terminal fragment of Efr3 (amino acids 340–782) can efficiently asso- ciate with Ypp1-GFP (Fig. 7 B). Second, as assessed by sub- cellular fractionation of yeast lysates Efr3-GFP was present in pellet fractions (20% in P13 and 80% in P100) similar to the PIK patch component Ypp1 (Fig. 6 A and Fig. 7 C). Third, Efr3- GFP localizes at PIK patch sites as determined by colocaliza- tion with Ypp1-mCherry (Fig. 7 D). Next, we addressed whether Efr3 functions in PtdIns4P synthesis and Stt4 PIK patch recruitment to the PM. For this, we generated a temperature-conditional mutant allele (see Materials and methods), efr3-1, which was unable to grow at the restrictive temperature of 38°C (Fig. 8 A). PtdIns4P levels were then determined at the nonpermissive temperature. Temperature-sensitive efr3-1 cells displayed reduced PtdIns4P levels as compared with wild-type EFR3 cells (Fig. 8 B). YPP1 AND EFR3 MEDIATE PIK PATCH ORGANIZATION • Baird et al. 1067