Figure 6. Stt4 stabilizes Ypp1 molecules on a membrane-bound fraction of the cell. (A) Sub- cellular fractionation of cells expressing HA-Ypp1 (left) in a wild-type background and HA-Ypp1 dis- tribution in the absence of SAC1 and STT4 (right). Protein component fractions in the pellet (P100; rcf of 100,000) or soluble (S100; soluble portion) fractions. Fractionation of G6PDH (soluble) and Pep13 (membrane-tethered Golgi component) are shown as controls. (B) FPLC elution proﬁle of HA- Ypp1 isolated from the S100 portion of sac1 stt4 cells (top). Fractions were collected at the indicated elution volumes, separated by SDS- PAGE, and detected using an anti-HA antibody. The molecular mass corresponding to each elu- tion is indicated below the blot. (C) Coimmuno- precipitation of HA-Ypp1 from yeast lysate that coexpressed GFP alone, GFP-Ypp1, or GFP-Stt4. (D) Coimmunoprecipitation of HA-Ypp1 with GFP- Ypp1 in the wild type, sac1 , and sac1 stt4 background. (E) FPLC sizing of E. coli–expressed and puriﬁed HIS6-Ypp1 using a Superdex 200 analytical column. The depressed PtdIns4P appears specific to the PM pool be- cause the PtdIns4P reporter GFP-2xPHOsh2 persists at the Golgi but is depleted from the PM in efr3-1 cells (Fig. 8 C). To assay the impact of Efr3 on PIK patch distribution, we placed EFR3 under the control of a doxycycline-repressible promoter (teto-EFR3) to selectively deplete expression of Efr3 in yeast har- boring a GFP-tagged PIK patch component, Stt4 or Ypp1. Treatment of the teto-EFR3 GFP-Stt4 strain with doxycycline for 12 h resulted in mislocalization of the lipid kinase to the diffuse cytoplasm (Fig. 8 D). In contrast to Stt4, Efr3-GFP lo- calization was still observed at the PM after depletion of Ypp1 in teto-YPP1 cells treated with doxycycline (Fig. 8 E). How- ever, the patch-like punctate distribution of Efr3-GFP at the PM was reduced (Fig. 8 E). In total, these results suggest that Efr3 mediates PIK patch localization to the PM but also imply that the Stt4–Ypp1 complex could control the clustering of proteins within the PIK patch. Discussion PIK patches are oligomeric lipid kinase complexes PIK patches are large protein complexes consisting of multiple copies of a lipid kinase along with other regulatory proteins oligomerized into a nonuniform punctate arrangement on their substrate membrane. In this work, we focused on PIK patches containing the essential PtdIns 4-kinase Stt4 and identified two essential components critical for Stt4 PIK patch assembly and localization. Previous work had already shown the Stt4 PIK patch to be separate from the PtdIns4P 5-kinase Mss4 PIK patch (Audhya and Emr, 2002) and we now demonstrate that it is also distinct from two other well known PM-associated complexes: actin patches and Pil1-based eisosomes. Kymographic mea- surements and FRAP experiments demonstrated that Stt4 PIK patches are static with limited exchange of molecules within the patch. These interesting properties coupled to the importance of Stt4-mediated PtdIns4P production at the PM prompted us to seek novel regulatory components of the Stt4 PIK patch. We un- covered two essential proteins. The first was Ypp1, a 95-kD TPR domain–containing protein whose role in the cell had yet to be clearly defined. The second was Efr3, an 89-kD putative membrane protein that shares homology with the Drosophila RBO protein. Our study determined that Ypp1 plays an essential role maintaining PtdIns4P levels at the PM by directly interacting with Stt4 to maintain PIK patch organization and stability of the PtdIns 4-kinase. First, we demonstrated that Ypp1 colocalizes and directly interacts with Stt4 at PIK patch sites on the PM. 1068 JCB • VOLUME 183 • NUMBER 6 • 2008