JCB: ARTICLE Assembly of the PtdIns 4-kinase Stt4 complex at the plasma membrane requires Ypp1 and Efr3 Dan Baird,1,2 Chris Stefan,1,2 Anjon Audhya,3 Sabine Weys,1,2 and Scott D. Emr1,2 1Weill Institute for Cell and Molecular Biology and 2Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853 3Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI 53706 Y G O L O I B L L E C F O L A N R U O J E H T T phate (PtdIns4P) is an essential signaling lipid that he phosphoinositide phosphatidylinositol 4-phos- regulates secretion and polarization of the actin cyto- skeleton. In Saccharomyces cerevisiae, the PtdIns 4-kinase Stt4 catalyzes the synthesis of PtdIns4P at the plasma membrane (PM). In this paper, we identify and character- ize two novel regulatory components of the Stt4 kinase complex, Ypp1 and Efr3. The essential gene YPP1 encodes a conserved protein that colocalizes with Stt4 at cortical punctate structures and regulates the stability of this lipid Introduction Cellular membranes are structured bilayers consisting of lipid and protein that define the boundaries of the cell and its organ- elles. Besides providing support and containment, membrane composition denotes organelle identity and transmits cellular signals. Among the various lipids incorporated into the mem- brane, inositol-containing phospholipids are ideally suited for transmitting signals because their inositol head group can be reversibly phosphorylated at three different hydroxyl positions. An array of kinases and phosphatases regulates phosphati- dylinositol phosphate (PIP) signaling by controlling inositol head group phosphorylation to efficiently mediate the levels and location of specific PIPs. Proteins are able to recognize these phosphorylated head groups through specific modular domains (e.g., PH, PX, FYVE, or ENTH, etc.) and transmit this informa- tion throughout the cell (Lemmon, 2003). Naturally, the impor- tance of PIP signaling is critical to a host of cellular processes ranging from receptor signaling pathways to regulation of actin cytoskeleton dynamics and vesicle trafficking (De Camilli et al., 1996; Odorizzi et al., 2000; Simonsen et al., 2001; Di Paolo and De Camilli, 2006). D. Baird and C. Stefan contributed equally to this paper. Correspondence to Scott D. Emr: sde26@cornell.edu Abbreviations used in this paper: FPLC, fast protein liquid chromatography; PIP, phosphatidylinositol phosphate; PIK, phosphoinositide kinase; PM, plasma mem- brane; PtdIns, phosphatidylinositol; PtdIns4P, phosphatidylinositol 4-phosphate; RBO, rolling blackout; TPR, tetratricopeptide repeat. The Rockefeller University Press $30.00 J. Cell Biol. Vol. 183 No. 6 1061–1074 www.jcb.org/cgi/doi/10.1083/jcb.200804003 kinase. Accordingly, Ypp1 interacts with distinct regions on Stt4 that are necessary for the assembly and recruit- ment of multiple copies of the kinase into phosphoinositide kinase (PIK) patches. We identify the membrane protein Efr3 as an additional component of Stt4 PIK patches. Efr3 is essential for assembly of both Ypp1 and Stt4 at PIK patches. We conclude that Ypp1 and Efr3 are required for the formation and architecture of Stt4 PIK patches and ultimately PM-based PtdIns4P signaling. The yeast Saccharomyces cerevisiae has proven useful as a genetic model system to study PIP signaling pathways be- cause several of the lipid kinases, lipid phosphatases, and ef- fectors are well conserved (Odorizzi et al., 2000). Each kinase or phosphatase localizes to a distinct region or organelle in the cell to control the levels and location of its corresponding phos- phoinositide. Stt4, the yeast homologue to the human type III phosphatidylinositol (PtdIns) 4-kinase PtdIns(4)KIII , is es- sential in yeast and localizes to punctate dots on the plasma membrane (PM; Audhya and Emr, 2002). The other essential yeast PtdIns 4-kinase, Pik1, localizes to the Golgi and acts in- dependently of Stt4 function (Hama et al., 1999; Walch-Solimena and Novick, 1999; Audhya et al., 2000; Strahl et al., 2005). The phosphatidylinositol 4-phosphate (PtdIns4P) generated by either of these kinases is turned over by the Sac1 domain– containing proteins Sac1, Sjl2, and Sjl3 (Guo et al., 1999; Foti et al., 2001). Among these three proteins, Sac1 has been dem- onstrated to be the PtdIns 4-phosphatase primarily responsible for metabolizing Stt4-generated PtdIns4P at the PM (Foti et al., 2001). Stt4 has been indirectly implicated in regulation of the cell cycle (Muhua et al., 1998), lipid metabolism (Voelker, 2005; Tabuchi et al., 2006), actin cytoskeleton dynamics, and activation © 2008 Baird et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the ﬁrst six months after the publica- tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). JCB 1061