FIG. 6.

Sec20p, Sec39p, and Dsl1p associate with peroxisomes. (A) A 20KgP fraction was prepared from wild-type BY4742 and mutant pex3Δ cells grown in oleic acid for 16 h for the isolation of peroxisomes by isopycnic gradient centrifugation on a discontinuous Nycodenz gradient as described in Materials and Methods. Equal volumes of each fraction were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted for the indicated proteins. P, peroxisome fractions; M, mitochondrial fractions. BY4742 cells coexpressing Pex3p-mRFP and Sec20p-GFP, Sec39p-GFP, or Dsl1p-GFP were grown in SCIM2 (B) for 4 h or in YEPD (C) for 16 h and fixed in 3.7% formaldehyde. Fluorescently tagged proteins were visualized for a single plane by confocal microscopy. Narrow arrowheads highlight areas of colocalization, and wide arrowheads highlight areas where Pex3p-mRFP is closely apposed to Sec20p-GFP, Sec39p-GFP, or Dsl1p-GFP. Bars, 5 μm. (D) BY4742 cells coexpressing Pex3p-GFP and Rtn1p-mRFP were grown in YEPD for 16 h. Cells were fixed with 3.7% formaldehyde, images were captured by confocal microscopy, and a maximum-intensity projection was created from a deconvolved 3D data set using Huygens software. Bar, 5 μm.