Figure 1. Ypp1 colocalizes with Stt4 at static cortical structures at the PM. (A) Cells expressing Ypp1-GFP were grown at 26°C and examined by ﬂuorescence microscopy. Cells shown are representative of over 200 cells observed. Arrows show regions of highly concentrated Ypp1-GFP. Bar, 4 μm. (B) Cells coexpressing GFP-Stt4 and Ypp1-mCherry were grown at 26°C and examined by confocal microscopy. As previously reported (Audhya and Emr, 2002), GFP-Stt4 localizes to cortical patches on the PM (top left) and with a similar distribution as Ypp1-mCherry (top right). An overlay of the two images (bottom left) shows overlap of the ﬂuorescent patches. Arrows indicate regions of the most conspicuous colocalization. Bar, 4 μm. (C) Schematic representation of Stt4, Ygr198w (Ypp1), and the Ypp1 human homologue TTC7a (available from GenBank/EMBL/DDBJ under accession no. NM_020458). Stt4 is a 215-kD protein with a lipid kinase unique domain (LKU) and a PtdIns 4-kinase domain (PI4K) as its only recognizable motifs. Ypp1 is a 95-kD protein encompassing multiple TPR domains and with conserved homologues in higher species. The schemat- ics are not to scale. (Kaksonen et al., 2005), GFP-Stt4 PIK patches are stable over the course of 3 min. To test whether the Stt4 molecules residing in these patches were also static instead of dynamically ex- changing, we performed FRAP on a region of the PM (Fig. 2 B and Video 2, available at http://www.jcb.org/cgi/content/full/ jcb.200804003/DC1). 3 min after photobleaching a PIK patch– rich region of the PM, none of the fluorescence had recovered. This is in stark contrast to the Stt4 product PtdIns4P as observed with a PtdIns4P reporter, GFP-2xPHOsh2 (Yu et al., 2004). After photobleaching a PtdIns4P-rich region of the PM, the fluores- cence of the GFP-2xPHOsh2 reporter recovers rapidly (Fig. 2 B and Video 3). Ypp1 directly interacts with Stt4 We next examined the contribution of Ypp1 on the interesting characteristics of Stt4 PIK patches. First, we determined if the two proteins interacted. A yeast strain expressing HA-tagged Ypp1 and either GFP-tagged Stt4 or GFP alone was detergent solubilized and immunoprecipitated using a GFP antibody. HA-Ypp1 showed no interaction with GFP alone, but a signifi- cant interaction with GFP-Stt4 (Fig. 2 C). Subsequent in vitro pulldown experiments using GST-tagged regions of Stt4 gener- ated from Escherichia coli identified at least two separate Ypp1- interacting regions. Both the N-terminal third of Stt4 (represented by amino acids 1–736) and a central region (represented by amino acids 736–1346) both bind full-length, E. coli–expressed HIS6-Ypp1 (Fig. 2 D). The C terminus of Stt4, which embodies the lipid kinase unique domain and the catalytic lipid kinase do- main, did not interact with Ypp1. Interestingly, there are no rec- ognizable structural motifs within the N terminus even though this region of Stt4 is critical for yeast viability; truncations lack- ing just the N-terminal 135 amino acids fail to localize to the PM or rescue stt4 cells (unpublished data). YPP1 and STT4 cooperate to positively maintain PtdIns4P levels Thus far, our data suggest that Stt4 and Ypp1 directly interact within the PIK patch. We next tested the role for Ypp1 in regu- lating the kinase activity of Stt4. Our first approach looked at the genetic interaction of mutant alleles of ypp1 in combination with the stt4-4 mutant (Audhya et al., 2000). We developed ypp1 temperature-conditional alleles with compromised growth at elevated temperatures. Seven clones of varying strength, ypp1-1–7, were selected based on the extent of their growth dis- parity at 26 and 37°C. These ypp1 mutant alleles were each car- ried on plasmids in the ypp1 background as described in the Materials and methods. Our current study utilizes the strongest of these alleles, ypp1-7, in combination with the stt4-4 mutant strain to detect synthetic growth defects. The ypp1-7 allele, which exhibited strong growth at 26°C, is dead at 37°C (Fig. 3 A). We dissected >20 tetrads generated from diploids with a deleted chromosomal copy of stt4 and ypp1 but carrying stt4-4 and ypp1-7 on plas- mids. We failed to isolate a single mutant strain with the stt4-4 ypp1-7 combination. The synthetic lethality of the combined mutant alleles suggests the two gene products function in paral- lel pathways or the same pathway. Additionally, we found that overexpression of YPP1 from a 2 plasmid could not rescue the growth defect of stt4-4 at 37°C (Fig. 3 A, bottom right); however, overexpression of the STT4 gene from a 2 plasmid mildly res- cues the growth defect of ypp1-7 at 37°C (Fig. 3 A, top right). We next addressed Ypp1’s effect on Stt4-mediated PtdIns4P synthesis by measuring PIP levels in yeast lacking the Ypp1 protein. Using the YPP1 clone from the previously char- acterized yTHC (yeast Tet-promoters Hughes Collection), we were able to significantly diminish Ypp1 levels (Hughes et al., 2000; Mnaimneh et al., 2004). The yTHC contains doxycycline- repressible clones from each of the essential yeast genes, allowing effective depletion of the corresponding protein over the course of around 12–24 h. After a 15-h incubation in the presence of YPP1 AND EFR3 MEDIATE PIK PATCH ORGANIZATION • Baird et al. 1063