FIG. 4.

Dependence of peroxisomal matrix protein import on direct Pex13-Pex14 interactions. Oleic acid-induced wild-type and pex13Δ cells expressing HA-Pex11 were subjected to double-immunofluorescence microscopy so as to localize HA-Pex11 in tandem with PTS1 protein Pcs60 (A), PTS2 protein Fox3 (B), or PTS1-like protein Cta1 (C). The same procedure was applied to pex13Δ cells expressing Pex13 (pKat113), Pex13_SH3 (pAS53), Pex13_loop (pAS71), and Pex13_loop+SH3 (pAS73). Detection was achieved with mouse monoclonal antibodies against the HA epitope combined with rabbit polyclonal antibodies against the individual matrix proteins. As secondary antibodies, Alexa Fluor 488-labeled anti-mouse IgG and Alexa Fluor 594-labeled anti-rabbit IgG were used. A congruent fluorescence pattern denotes colocalization of HA-Pex11 with the analyzed matrix proteins, a finding which is readily revealed in the merged images. (D) From the same cells, a postnuclear supernatant (PNS) was produced and subfractionated into a 25,000 × g pellet fraction (P) enriched for peroxisomes and a supernatant fraction (SN) enriched for the cytosol. Equivalent volumes of fractions were loaded on the gels, transferred to nitrocellulose membranes, and analyzed for the distributions of the specified proteins.