S1Supplemental Data A Novel Transport Pathway for a Yeast Plasma Membrane Protein Encoded by a Localized mRNA

TGG-3. The resulting product was cloned between the SpeI andMedia and Yeast Strains XhoI sites of pPS1890.Media were prepared as described previously [S1]. Yeast transfor-The plasmid pCJ022 encodes Ist2p on a CEN plasmid. It was con-mation was carried out according to the method of Gietz et al. [S2]. structed by amplifying IST2 including 318 nucleotides of its 5-UTRAll strains used in this study (Table S1) were isogenic derivatives and 996 nucleotides of its 3-UTR with primers 5- CGGGATCCACof W303 [S3] whose full genotype is MATa, ade2-1, can1-100, his3-CACTACATCAGTGTG-3 and 5-ACTCGAGACCCTATCGAATGAA11,15, leu2-3,112, trp1-1, ura3-1, GAL, psi. GAGTGC-3. The resulting product was cloned between the BamHIsec- and myo2-66 mutants were provided by P. Novick and R.A. and XhoI sites of pRS316 [S5].Singer, respectively, and crossed into the W303 background. Fluorescence MicroscopyConstruction of Plasmids Yeast cells expressing YFP-Ist2p and Hxt1p-CFP under control ofThe plasmid pCJ083 encodes GFP-Ist2p under its own promoter the GAL1 promoter were grown at 25C in YEP medium with 20 g/l for integration into the LEU2 locus. It was constructed by amplifying raffinose and 60 mg/l adenine to early logarithmic phase. Expression 597 nucleotides of the IST2-5UTR with the primers 5-GCTCTA was induced by the addition of galactose to a final concentratio