Figure 2. Pex3Bp is a peroxisomal... | Rockefeller University Press

Figure 2.

$Figure 2. Pex3Bp is a peroxisomal integral membrane protein. (A) Pex3Bp-mRFP colocalizes with the peroxisomal chimeric reporter Pot1p-GFP to punctate structures characteristic of peroxisomes by confocal microscopy. The right panel presents the merged image of the left and middle panels, with colocalization of Pex3Bp-mRFP and Pot1p-GFP shown in yellow. Bar, 5 µm. (B) Pex3Bp-mRFP localizes to the 20KgP subcellular fraction enriched for peroxisomes. Immunoblot analysis of equivalent portions of the PNS, 20KgP, and 20KgS subcellular fractions from cells expressing Pex3Bp-mRFP was performed with antibodies to mRFP and to the peroxisomal matrix enzyme thiolase (Pot1p). (C) Pex3Bp exhibits the characteristics of an integral membrane protein. The 20KgP fraction from cells expressing Pex3Bp-mRFP was treated with Ti8 buffer to lyse peroxisomes and then subjected to centrifugation to yield a supernatant fraction (Ti8S) enriched for matrix proteins and a pellet fraction (Ti8P) enriched for membrane proteins. The Ti8P fraction was further treated with alkali Na2CO3 and separated by centrifugation into a supernatant fraction (CO3S) enriched for peripheral membrane proteins and a pellet fraction (CO3P) enriched for integral membrane proteins. Equivalent portions of each fraction were analyzed by immunoblotting. Immunodetection of Pot1p and Pex2p marked the fractionation profiles of a peroxisomal matrix and integral membrane protein, respectively. (D) pex3BΔ cells exhibit slightly retarded growth on oleic acid medium. Cells of the wild-type strain E122 and of the deletion strains pex3BΔ and pex3Δ were grown to mid-log phase in liquid YPD, incubated in liquid YPBO for 1 d, spotted at dilutions of 10−1–10−4 on YPBO agar, and grown for 2 d at 30°C.$

Pex3Bp is a peroxisomal integral membrane protein. (A) Pex3Bp-mRFP colocalizes with the peroxisomal chimeric reporter Pot1p-GFP to punctate structures characteristic of peroxisomes by confocal microscopy. The right panel presents the merged image of the left and middle panels, with colocalization of Pex3Bp-mRFP and Pot1p-GFP shown in yellow. Bar, 5 µm. (B) Pex3Bp-mRFP localizes to the 20KgP subcellular fraction enriched for peroxisomes. Immunoblot analysis of equivalent portions of the PNS, 20KgP, and 20KgS subcellular fractions from cells expressing Pex3Bp-mRFP was performed with antibodies to mRFP and to the peroxisomal matrix enzyme thiolase (Pot1p). (C) Pex3Bp exhibits the characteristics of an integral membrane protein. The 20KgP fraction from cells expressing Pex3Bp-mRFP was treated with Ti8 buffer to lyse peroxisomes and then subjected to centrifugation to yield a supernatant fraction (Ti8S) enriched for matrix proteins and a pellet fraction (Ti8P) enriched for membrane proteins. The Ti8P fraction was further treated with alkali Na₂CO₃ and separated by centrifugation into a supernatant fraction (CO₃S) enriched for peripheral 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. (D) pex3BΔ cells exhibit slightly retarded growth on oleic acid medium. Cells of the wild-type strain E122 and of the deletion strains pex3BΔ and pex3Δ were grown to mid-log phase in liquid YPD, incubated in liquid YPBO for 1 d, spotted at dilutions of 10⁻¹–10⁻⁴ on YPBO agar, and grown for 2 d at 30°C.