FIG. 5.

YlInp1p is a peripheral membrane protein of peroxisomes. (A) YlInp1p-GFP colocalizes with mRFP-SKL to punctate structures characteristic of peroxisomes by direct confocal microscopy. The right panel presents the merged image of the left and middle panels, with colocalization of YlInp1p-GFP and mRFP-SKL shown in yellow. Bar, 2 μm. (B) Immunoblot analysis of whole-cell lysates of the wild-type strain E122 and the deletion strain Ylinp1Δ probed with anti-YlInp1p antibodies. Strains were incubated in YPBO for 9 h. Arrowheads point to a nonspecific immunoreactive polypeptide present in the lysates of both E122 and Ylinp1Δ cells. (C) YlInp1p localizes to the 20KgP subcellular fraction enriched for peroxisomes. Immunoblot analysis of equivalent portions of the 20KgS and 20KgP subcellular fractions from wild-type E122 cells was performed with antibodies to YlInp1p and to the peroxisomal matrix enzyme thiolase (Pot1p). (D) YlInp1p cofractionates with peroxisomes. Organelles in the 20KgP fraction were separated by isopycnic centrifugation on a discontinuous sucrose gradient. Fractions were collected from the bottom of the gradient, and equal portions of each fraction were analyzed by immunoblotting. Fractions enriched for peroxisomes and mitochondria were identified by immunodetection of Pot1p and Sdh2p, respectively. (E) Purified peroxisomes were ruptured by treatment with Ti8 buffer and subjected to ultracentrifugation to obtain a supernatant fraction, Ti8S, enriched for matrix proteins and a pellet fraction, Ti8P, enriched for membrane proteins. The Ti8P fraction was treated further with alkali Na₂CO₃ and separated by ultracentrifugation into a supernatant fraction (CO₃S) enriched for peripherally associated membrane proteins and a pellet fraction (CO₃P) enriched for integral membrane proteins. Equivalent portions of each fraction were analyzed by immunoblotting. Immunodetection of Pot1p and Pex2p marked the fractionation profiles of a peroxisomal matrix and integral membrane protein, respectively.