of a MAPK pathway (Audhya and Emr, 2002). Despite the obvious importance for Stt4 and its lipid product, PtdIns4P, the precise regulation of Stt4 activity at the PM remains poorly understood. This study describes the analysis of a unique cortical structure, Stt4-containing phosphoinositide kinase (PIK) patches. Previous work has demonstrated that both the PM-localized lipid kinases, Stt4 and Mss4, cluster into distinct cortical struc- tures (Audhya and Emr, 2002). Furthermore, mammalian lipid kinases often localize to patches at sites of receptor-mediated signaling or membrane attachment to the extracellular matrix (Honda et al., 1999; Ling et al., 2003; Heck et al., 2007). Although PIK patches seem to be a widely adopted structure, it is unclear why cells need to maintain these arrangements. In this paper, we focus on Stt4 PIK patches and anticipate that com- mon themes regarding these structures may emerge. We identify two novel components of the Stt4 PIK patch: Ypp1 and Efr3. Ypp1 is an essential protein that directly con- tacts distinct sites on Stt4 to organize and stabilize PIK patches at the PM. Ypp1 and Stt4 colocalize on the PM to form the core of a large Stt4 complex visualized as punctate patches. Loss of Ypp1 from this complex mislocalizes Stt4 and results in the subsequent degradation of the lipid kinase. Efr3 likewise local- izes with Ypp1 at Stt4 PIK patches, physically interacts with Ypp1, and is necessary for assembly of the PIK patch compo- nents Stt4 and Ypp1 at the PM. Consequently, cells lacking either Ypp1 or Efr3 fail to assemble the Stt4 PIK patch and fail to maintain proper PtdIns4P levels. Results Initial characterization of the Stt4 PIK patch Using a three-dimensional reconstruction of z stacks from cells expressing an N-terminally tagged GFP-STT4 construct, we have been able to estimate the number of Stt4 PIK patches on the PM of an individual cell as shown in Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200804003/DC1). The three-dimensional reconstruction of the cells is a representative sample of several previously analyzed populations. The fluor- escence data indicate that each cell has 20–25 regions of dense fluorescence corresponding to a cluster of GFP-Stt4 within the PIK patch. Additionally, coimmunoprecipitation of HA-Stt4 with GFP-Stt4 from cells coexpressing the two epitope– tagged Stt4 proteins demonstrates that these proteins form a physical interaction (Fig. S1 A). Therefore, we believe the dense fluorescent cluster of GFP-Stt4 represents an oligomer of Stt4 at the PIK patch site. With the understanding that roughly 850 Stt4 molecules populate the average cell (Ghaemmaghami et al., 2003) and that the plasmid-expressed GFP-Stt4 fusion is main- tained at the same concentration as wild-type Stt4 (Audhya and Emr, 2002), we can estimate that each Stt4 PIK patch contains 30 Stt4 molecules. This molecular abundance of Stt4 at a PIK patch corroborates well with the fluorescence intensity of a PIK patch measured against a known standard (Fig. S1 B). Thus, these results suggest that multiple copies of Stt4 oligo- merize at PIK patches. Identiﬁcation of a novel component of the Stt4 PIK patch To understand Stt4 PIK patch assembly and regulation, we sought novel Stt4-interacting partners. We examined several candidate PtdIns 4-kinase regulators by experimentally testing the various Stt4-interacting proteins cataloged in the Saccharo- myces Genome Database and identified one uncharacterized essential protein: YGR198w (recently named Ypp1 [Flower et al., 2007]). This interaction was originally published by Hazbun et al. (2003), who isolated Ypp1 and corresponding binding partners through tandem affinity purification and subse- quent mass spectrometry to identify Stt4 as the primary binding partner. To determine the localization of Ypp1 in live yeast cells, the sequence encoding GFP was integrated at the 3 end of Ypp1’s chromosomal locus to generate Ypp1-GFP (Fig. 1 A). Similar to GFP-Stt4 (Audhya and Emr, 2002), Ypp1-GFP local- ized to several punctate structures on the periphery of the cell with a majority of these patches concentrated in the mother cell. Furthermore, strong colocalization of Ypp1-mCherry with GFP- Stt4 on these punctate patches suggests they reside at the same cortical structure on the PM (Fig. 1 B). Primary sequence analysis of Ypp1 reveals two separate tetratricopeptide repeat (TPR) domains as its only recognizable structural motif. TPR domains exist in nearly all organisms and have been shown to mediate a variety of protein–protein inter- actions (for review see D’Andrea and Regan, 2003). Further- more, YPP1 homologues are evident in several species; in humans, the closest homologue is TTC7a (TPR domain 7A; Fig. 1 C). Despite their similar sequence structure, however, TTC7a is not able to rescue the inviability of ypp1 yeast (un- published data). Stt4–Ypp1 PIK patches are static structures separate from other PM-based components Stt4–Ypp1 PIK patches have been shown to be separate struc- tures from Mss4 PIK patches (Audhya and Emr, 2002). How- ever, it has been hypothesized that the Ypp1 PM structures are cortical actin patches because of their similar distribution (Flower et al., 2007). This seems unlikely as we found no signif- icant colocalization of Abp1–monomeric RFP, a known compo- nent of cortical actin structures enriched in the bud (Stefan et al., 2005), with Ypp1-GFP or GFP-Stt4. Also, treatment with la- trunculin A has no effect on the localization or dynamics of Stt4–Ypp1 PIK patches (Fig. S2 A, available at http://www.jcb .org/cgi/content/full/jcb.200804003/DC1). We examined whether the distribution of Stt4–Ypp1 PIK patches show similarity to another PM localized structure: Pil1-based eisosomes (Walther et al., 2006). Pil1-mCherry and GFP-Ypp1 show very limited colocalization (Fig. S2 B). Furthermore, GFP-Ypp1 localizes normally in pil1 cells (unpublished data). These findings show that Stt4 PIK patches and eisosomes are distinct organizational units of the PM. Subsequently, we studied the dynamics of Stt4 and Ypp1 at PIK patches and compared it to the Stt4 product PtdIns4P. Fig. 2 A is a fluorescent image with the corresponding kymo- graph of GFP-Stt4 and Ypp1-GFP. Unlike cortical actin patches 1062 JCB • VOLUME 183 • NUMBER 6 • 2008