4.1 (Intelligent Imaging Innovations). The contrast and brightness of the images were further adjusted in Photoshop (Adobe) to give the clearest presentation of the results. Fluorescence intensity of the GFP (FITC) channel of unprocessed JPEG images was quantiﬁed using ImageJ 1.38× (National Institutes of Health). Plot proﬁles were acquired by drawing a line around the perime- ter of the PM and the ﬂuorescence intensity values were exported to Excel 2004 (Microsoft) and normalized. Pixel-by-pixel analysis was performed using Softworx Data Inspector (Applied Precision, LLC). The Fluorescence Intensity (IF) of LacI-GFP was measured using a 5 × 5-pixel region of interest (ROI) and subtracted against the nuclear background (5 × 5-pixel ROI of LacI-GFP not present in the dot; Fig. S1 B). IF of GFP-Stt4 was measured in whole cells using a 5 × 5-pixel ROI and subtracted against background (Fig. S1 B). In vivo analysis of phosphoinositides Phosphoinositide levels were analyzed as previously described (Rudge et al., 2004). For quantitative analyses, dried pellets were resuspended in sterile water and 107 cpm of sample was separated on a Partisphere SAX column (GE Healthcare) attached to an HPLC system (Shimadzu Manufacturing) and a 610TR on-line radiomatic detector (PerkinElmer) using Ultima Flo scintillation ﬂuid (PerkinElmer). The HPLC and on-line detector were con- trolled with EZStart 7.2.1 (Shimadzu) and ProFSA 3.3 (PerkinElmer) soft- ware, respectively, with ﬁnal data analysis taking place in the latter. Subcellular fractionation 20 A600 equivalents of yeast cells were spheroplasted and lysed in 4.3 ml of ice-cold Tris Lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 200 mM sorbitol) containing 0.1 mM AEBSF, 0.1 mM chymostatin, 1 μM pep- statin A, and protease inhibitor cocktail (Complete EDTA-free; Roche). 1-ml aliquots of lysates were precleared for 5 min at 500 g and were then cen- trifuged at 13,000 g for 10 min at 4°C to generate pellet (P13) and super- natant (S13) fractions. The 1-ml S13 fraction was centrifuged at 100,000 g for 1 h at 4°C in an ultracentrifuge (TL-100; Beckman Coulter) to generate the P100 and S100 fractions. Fractions were resolved on a 6% SDS-PAGE gel and immunoblotted. Protein expression and puriﬁcation BL21(DE3)pLys cells (EMD) transformed with protein expression plasmids were grown at 37°C to an A600 of 0.4. The bacteria were then shifted to 20°C for 1 h and protein expression was induced by the addition of IPTG to 100 μM for 12 h. For cells expressing GST fusion proteins, cell pellets were resuspended in buffer A (20 mM Hepes, pH 8.0, 250 mM NaCl, 1 mM EDTA 0.5% Tween 20, and 1 mM NaN3) containing protease inhibitor cocktail and lysed by sonication followed by French Press. Lysates were gently cleared (45 min at 800 g) and GST-tagged proteins were puriﬁed from the supernatant using glutathione Sepharose 4B (GE Healthcare). After 2 h of incubation, the bead/lysate mix was washed in buffer A three times, and then protein was eluted from the beads with buffer A and 10 mM of reduced glutathione. Proteins were subsequently concentrated and dia- lyzed into buffer A. For cells expressing HIS6 fusion proteins, cell pellets were resus- pended in buffer B (20 mM Tris, pH 7.5, 500 mM NaCl, and 20 mM imid- azole) with protease inhibitors and prepared similar to GST fusion puriﬁcation (previous paragraph) except that lysates were cleared by centrifugation (45 min at 13,000 g) and puriﬁed with Ni-NTA agarose (QIAGEN). The beads were washed extensively and the protein was eluted with buffer B plus 200 mM imidazole. Sizing of HA-Ypp1 (Fig. 6 B) and HIS6-Ypp1 (Fig. 6 E) was performed on Superdex 300 and 200 analytical columns (GE Healthcare), respectively. Regulation of gene transcription with doxycycline Strains from the yTHC (Open Biosystems) were used to regulate expression of essential genes. Typically, the strain of interest was grown up in synthetic media and then diluted to an OD600 of 0.01, at which time doxycycline was added to a ﬁnal concentration of 20 μg/ml. Subsequently, yeast were grown for 12 h and the abundance of the protein of interest was then measured to determine the relative amount remaining in the cells. Condi- tions varied slightly between strains tested. Online supplemental material Fig. S1 shows coimmunoprecipitation of HA-Stt4 with GFP-Stt4 from yeast lysates and quantiﬁcation of GFP-Stt4 abundance in PIK patches compared with a LacI-GFP standard. Fig. S2 shows the localization of GFP-Ypp1 com- pared with other well characterized PM structures. Fig. S3 shows colocal- ization of Psr1-28-GFP-Ypp1 with mCherry-Stt4. Video 1 is a three-dimensional reconstruction of z stacks from cells expressing GFP-Stt4. Video 2 shows FRAP of GFP-Stt4–based PIK patches. Video 3 shows FRAP of the PtdIns4P reporter GFP-2xPHOsh2. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200804003/DC1. We are grateful to Jason Yamada-Hanff for excellent technical support through the course of this study. We thank Dr. David Teis for extensive microscopy as- sistance and critical reading of the manuscript and Dr. Sarah Rue for sharing unpublished plasmids. S.D. Emr is supported by the Weill Institute for Cell and Molecular Biol- ogy at Cornell University. The project described was supported by grant F32GM084567 from the National Institute of General Medical Sciences. 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