Figure 1.

Per1p is mainly localized in the ER. (A) Tagged Per1 proteins are functional and complement the CFW sensitivity and the temperature sensitivity of per1Δ cells. Isogenic per1Δ cells carrying the pRS316T (vector), pMF917 (PER1), pMF918 (PER1-HA), and pMF921 (PER1-mRFP) were spotted on YPAD or YPAD plates containing 7.5 μg/ml CFW after making a 5× serially diluted solution and then incubating for 2 d at 30 or 37°C. (B) Localization of Per1-mRFP. per1Δ cells carrying pMF921 were visualized by fluorescence microscopy. DIC, differential interference contrast. (C) Fractionation of Per1-HA. Exponentially growing per1Δ cells carrying pMF918 were converted to spheroplasts, homogenized, and subjected to the following series of centrifugations: 300 × g for 5 min, 13,000 × g for 15 min, and 100,000 × g for 60 min. Aliquots were taken from the 13,000 × g pellet (P13) and the 100,000 × g pellet (P100) and supernatant (S100) and then analyzed by immunoblotting.