Figure 9. Organization of Stt4 PIK patches at the PM. Ypp1 is a multivalent linker of Stt4 mol- ecules ensuring the stability of the lipid kinase and the organization of the PIK patch. Efr3 anchors the Stt4–Ypp1 complex at the PM by binding Ypp1. TMD, transmembrane domain; LKUD, lipid kinase unique domain; PI4K, PtdIns 4-kinase domain. Efr3, the yeast homologue of Drosophila RBO protein, acts as a PIK patch membrane anchor We also identified Efr3, the yeast homologue of Drosophila RBO, as a critical component to maintain PIK patches at the PM. RBO is a multipass transmembrane protein localized to the PM with an essential role in synaptic vesicle trafficking. Based on Efr3’s capability to localize to the PM in the absence of other PIK patch components, Efr3 defined a candidate membrane an- chor for Stt4 PIK patches. In support of this, a hydrophobicity diagram generated from TMpred software predicts Efr3 to be a one- or two-pass transmembrane protein. Therefore, we propose that Efr3 recruits and anchors the Stt4 PIK patch to the PM. Placing Efr3 in the PM would allow the protein to relay extracel- lular or membrane activities directly to the Stt4 PIK patch to mediate PtdIns4P metabolism. This idea is supported by the fact that Stt4 activity is important for maintaining cell wall integrity, and Efr3, therefore, may provide a link to the cell wall (Yoshida et al., 1994; Audhya et al., 2000; Audhya and Emr, 2002). Future work will focus on how changes in the extracellular environment or within the membrane, such as cell stress or damage condi- tions, impact Stt4 PIK patch dynamics and kinase activity. Can the known function of RBO provide further insight into Efr3’s role in the Stt4 PIK patch? RBO has been shown to be important for synaptic vesicle exocytosis based on the obser- vation that synaptic vesicles accumulate at the presynaptic active zone in rbots mutant neurons shifted to the nonpermissive temperature. The authors attributed the phenotype to a disrup- tion in the balance between synaptic vesicle consumption (exo- cytosis) and recycling (endocytosis), but favored the idea that RBO functions at the post-docking step of synaptic vesicle exo- cytosis (Huang et al., 2006). Appropriately, many of the steps involved in synaptic vesicle exocytosis are regulated by lipid composition at the active zone (including the PIP2 binding pro- teins synaptotagmin and CAPS) and provide a potential connec- tion to our findings on Efr3’s role in PIP dynamics (Osborne et al., 2006). Previous studies have indicated a link between the secre- tory and cell polarity pathways in yeast (Aronov and Gerst, 2004; Pruyne et al., 2004) and both Stt4 and Mss4 activity have been shown to be required for cell polarity through the actin cyto- skeleton (Audhya et al., 2000; Audhya and Emr, 2002). Besides impacting secretion through actin cytoskeleton dynamics, Stt4 PIK patches may enhance secretion by regulating lipids neces- sary for exocyst-dependent vesicle fusion. For example, studies have suggested that polarized cell growth is dependent on the exocyst complex through the PtdIns(4,5)P2 binding components Exo70 and Sec3. Disrupting the PIP binding domain of Exo70 or Sec3 mislocalizes the exocyst complex from the PM and se- verely impacts secretion (He et al., 2007; Zhang et al., 2008). Future work will investigate potential links between Efr3 activ- ity at the PIK patch and polarized secretion. A model for PIK patches and their biological function Fig. 9 provides a model that illustrates how we propose the Stt4 PIK patch organizes at the PM. First, Ypp1 is capable of inter- acting with at least two separate regions within the N terminus of Stt4 (Fig. 2 D). As a result, Ypp1 could act as a multivalent linker to organize/cluster multiple ( 25–30) Stt4 proteins into a PIK patch. Stt4–Ypp1 complexes localize to the PM and fur- ther assemble through Efr3 to direct Stt4-mediated PtdIns4P production at the PM. Additional components, such as Sfk1 (Audhya and Emr, 2002), may contribute to the organization and activation of this complex. Because there are potentially other factors associated with regulation of this complex, our future work will be directed toward identifying these components and determining how each factor mediates PIK patch dynamics and PtdIns4P metabolism. What is the biological significance of forming a large, static Stt4 PIK patch at the PM? Placing multiple PtdIns kinase enzymes into a common complex could permit coordinate acti- vation/inactivation of all the kinases in the complex (PIK patch). This would allow the cell to regulate local levels of PtdIns4P in the PM (activation of some PIK patches while retaining others inactive). However, further experiments are needed to test this idea. Alternatively, the stability of the Stt4 PIK patch suggests they may reside at membrane contact sites linked to the ER. YPP1 AND EFR3 MEDIATE PIK PATCH ORGANIZATION • Baird et al. 1071