Mislocalization of peroxisomal proteins by pex19Δ mutants. Oleate-grown (A) and methanol-grown (B) spheroplasts of wild-type and pex19Δ cells (SKF14 and SMD1163) were lysed and subjected to sequential differential centrifugation. Equivalent amount of the PNS, 27,000 × g supernatant (S27), 27,000 × g pellet (P27), 100,000 × g supernatant (S100), and 100,000 × g pellet (P100) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with the indicated antibodies. (C) The PNS from methanol-grown cells was adjusted to 60% sucrose, layered with 55 and 35% sucrose, and centrifuged to allow floatation of membranes into the lighter fractions as described in MATERIALS AND METHODS. P, pelleted material at the bottom of the tube. Fractions are numbered from the bottom to the top and were collected as described in MATERIALS AND METHODS. Fractions 1 and 2 are the 60% sucrose load zone; fractions 3 and 4 are the 55% sucrose float zone. The top fraction contained no protein.