Table I. Yeast strains used in this study Strain BY4741 BY4742 DBY4 DBY16 DBY21 DBY25 DBY37 DBY42 DBY45 DBY47 DBY48 DBY72 DBY74 DBY76 DBY117 DBY131 DBY134 DBY294 DBY298 DBY417 JBY404 R1158 TH2102 TH6139 DBY454 DBY482 YCS443 DBY470 Genotype MATa his3 1 leu2 0 met15 0 ura3 0 MAT his3 1 leu2 0 lys2 0 ura3 0 BY4741; YPP1-GFP::HIS3MX BY4741; ypp1 ::HIS3MX6 carrying pRS415ypp1-7 (LEU2 CEN6 ypp1-7) BY4741; ypp1 ::HIS3MX6 carrying pSR54YPP1 (LEU2 CEN6 YPP1) BY4741; ypp1 ::HIS3MX6 carrying pBP73-CYPP1 (URA3 CEN6 YPP1) BY4741; ypp1 ::HIS3MX6 sac1 ::KanMX BY4741; stt4 ::HIS3MX6 carrying pRS416GFP-STT4 (URA3 CEN6 GFP-STT4) BY4741; stt4 ::HIS3MX6 carrying pRS415stt4-4 (LEU2 CEN6 stt4-4) BY4741; sac1 ::KanMX DBY21; stt4 ::HIS3MX6 carrying pRS415stt4-4 (LEU2 CEN6 stt4-4) DBY10; stt4 ::HIS3MX6 carrying pRS415stt4-4 (LEU2 CEN6 stt4-4) DBY42; YPP1-mCherry::HIS3MX6 TH2102; stt4 ::HIS3MX6 carrying pRS416GFP-STT4 (URA3 CEN6 GFP-STT4) BY4741; pik1 ::HIS3MX6 carrying pRS315pik1-83 (LEU2 CEN6 pik1-83) DBY47; stt4 ::HIS3MX6 TH2102; YPP1-HA::HIS3MX6 DBY4; ABP1-mRFP::HIS3MX6 DBY4; PIL1-mCherry::HIS3MX6 BY4741; stt4 ::HIS3MX6 carrying pSR54mCherry-Stt4 (LEU2 CEN6 mCherry- Stt4) and pRS416Psr1-28-GFP-YPP1 (URA3 CEN6 Psr1-28-GFP-YPP1) ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 opi1 ::LEU2 SEC63-13myc::KANr INO1:LacO128:URA HIS3:LacI-GFP BY4741; URA3::CMV-tTA R1158; pYGR198w::kanR-tet07-TATA R1158; pSTT4::kanR-tet07-TATA DBY74; EFR3-GFP::HISMX3 DBY42; efr3 ::HISMX3 carrying pCM187EFR3 (URA teto EFR3) efr3 ::HISMX3 carrying pRS415efr3-1 (LEU CEN6 efr3-1) TH2102; EFR3-GFP::HISMX3 Reference or source Mnaimneh et al., 2004 Mnaimneh et al., 2004 This study This study This study This study This study This study; Audhya and Emr, 2002 This study; Audhya and Emr, 2002 Winzeler et al., 1999 This study This study This study This study This study This study This study This study This study This study Brickner and Walter, 2004 Hughes et al., 2000 Peng et al., 2003 Peng et al., 2003 This study This study This study This study These contact sites have been hypothesized to be regions through which lipids can easily traffic between closely apposed mem- branes/organelles (Levine and Loewen, 2006). There is some evi- dence to support this notion. First, Sac1, the PIP phosphatase that antagonizes Stt4 activity by turning over PtdIns4P at the PM, is an integral transmembrane protein anchored in the ER (Foti et al., 2001). Second, proteomics and genetic screens have identified several potential interactions between Stt4 and ER components (Gavin et al., 2002; Tabuchi et al., 2006). There- fore, Stt4 PIK patch components may be spatially restricted on the PM to sites that are juxtaposed with the ER and these sites could mediate local PIP production and signaling between the two membranes. Future work will explore the possibility that Stt4 PIK patches reside at these potential membrane contact sites and may determine the mechanism for formation and regu- lation of these contact sites. Materials and methods Yeast strains and plasmid construction A list of all S. cerevisiae strains and plasmids used in this study and their genotypes can be found in Table I. The Longtine method (Longtine et al., 1998) was used to introduce gene deletions and epitopes into yeast by homologous recombination. The cloning vectors pRS415 and pRS416 are described in Sikorski and Hieter (1989). The ﬂuorescent fusion vector pBP73-C was a gift from W. Parrish (The Feinstein Institute for Medical Research, Manhasset, NY). The bacterial expression vector pGEX6P-1 used for all the GST constructs is from GE Healthcare and the pET-23d+ vector used for all the HIS6 constructs is from EMD. The cloning regions of all plasmids were completely sequenced to ensure that no mutations were introduced in the cloning process. The GFP-STT4 fusion plasmid has been described previously (Audhya and Emr, 2002). The pRS315pik1-83 plasmid was subcloned from pRS314pik1-83 (a gift from J. Thorner, University of California, Berkeley, Berkeley, CA; Hendricks et al., 1999). The pRS415stt4-4 plasmid has been described previously (Audhya et al., 2000). Generation of temperature-sensitive ypp1 and efr3 alleles To construct adequate alleles, we used PCR-mediated random mutagene- sis of pRS415YPP1 to generate mutant ypp1. These plasmids were subse- quently transformed into haploid yeast lacking the chromosomal copy of YPP1 but carrying a URA3+ pRS416YPP1 plasmid. Mutagenized plas- mids were selected on 5-FOA plates and then the relative growth capa- bilities of the various clones were measured at 26 and 37°C. A similar procedure was also used to generate the efr3-1 temperature-sensitive mu- tant allele. Fluorescence microscopy and quantiﬁcation of ﬂuorescence intensity Living cells expressing ﬂuorescent fusion proteins were grown in minimal media to an A600 of 0.4–0.6. Fluorescence microscopy was performed at 26°C (unless otherwise indicated) in synthetic media. Most images were captured with a microscopy system (DeltaVision RT; Applied Precision, LLC) equipped with a microscope (IX71; Olympus), a PlanApo 100× ob- jective (1.35 NA; Olympus), FITC and rhodamine ﬁlters, and a digital camera (Cool Snap HQ; Photometrics) and were deconvolved using soft- WoRx 3.5.0 software (Applied Precision, LLC). Images for Fig. 1 B and Fig. 2 B were collected with a spinning disc confocal microscopy system (3I; Yokogawa) using a microscope (DMI3000B; Leica) equipped with FITC and rhodamine ﬁlters, a 63× (1.40 NA) objective, and a digital camera (QuantEM; Photometrics) and were deconvolved using Slidebook 1072 JCB • VOLUME 183 • NUMBER 6 • 2008