FIG. 1.
The peroxisomal matrix protein Pot1p is mislocalized in cells repressed for SEC20 and SEC39 expression. (A) Wild-type strain R1158 and strains THC-SEC7, THC-SEC61, THC-SEC20, and THC-SEC39 harboring genomic POT1-GFP were incubated for 18 h in YEPD, at which time cells were transferred to oleic acid-containing medium (YPBO) for an additional 90 min to induce the expression of Pot1p-GFP. (B) Pot1p localizes to peroxisomes in repressed THC-SEC11, THC-SEC13, THC-SEC17, THC-SEC18, THC-SEC65, and THC-COPI cells. The indicated THC strains expressing genomic POT1-GFP were incubated in YEPD as for panel A and then transferred to YPBO medium for 2 h (SEC13) or 4 h (remaining strains). Repression of essential SEC genes under the control of the TetO7 promoter in panels A and B was achieved by addition of 10 μg doxycycline/ml to the medium. The localization of Pot1p-GFP to peroxisomes was monitored by epifluorescence microscopy. Bars, 5 μm. (C) Sec20p levels are reduced in doxycycline-treated or untreated THC-SEC20 cells. Equal amounts of protein from whole-cell lysates of doxycycline-treated or untreated THC-SEC20 cells and untreated wild-type R1158 cells were subjected to immunoblot analysis with antibodies to Sec20p. Immunodetection of Act1p was used as a control for protein loading.