Fig. 1. PTS2 protein mislocalization in Hppex20 cells. (A) Post nuclear supernatants prepared from glucose/methylamine-grown wild type (WT) and Hppex20 cells were subjected to differential centrifugation. P3-30,000 g pellet, S3-30,000 g supernatant. Western blots were probed with antibodies against several H. polymorpha proteins: AMO (PTS2 matrix protein); porin (mitochondria); ADH-alcohol dehydrogenase (cytosol) and Pex14p (peroxisomal membrane). Equal portions of each fraction were loaded per lane. (B) Fluorescence microscopy of WT and Hppex20 cells expressing a fusion protein consisting of the first 50 amino acids of S. cerevisiae thiolase and GFP and DsRed containing the PTS1 sequence –SKL. In WT cells, green and red fluorescence is present in spots, indicative of peroxisomes. In pex20 cells, green fluorescence is observed in the cytosol, whereas red fluorescence is localized in spots.