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Fig. 5. A region of Pex19p encompassing amino acid residues 12-73 and 40-131 is sufficient for binding to Pex3p. (A) HA₂- or Myc₆-HA₂-tagged Pex19p truncation mutants were co-expressed with FL-Pex3p-EGFP in COS7 cells. HA₂-Pex19p and Myc₆-HA₂-Pex19p variants were immunoprecipitated using rabbit anti-HA antibody and analyzed by SDS-PAGE. HA₂-Pex19p and Myc₆-HA₂-Pex19p proteins were detected by immunoblot with mouse anti-HA antibody (lower panel). FL-Pex3p-EGFP co-immunoprecipitated with Pex19p was detected with anti-GFP antibody (upper panel). Lanes 1-10, 10% input used for immunoprecipitation; lanes 11-20, immunoprecipitates. Solid arrowhead indicates FL-Pex3p-EGFP and open arrowheads, HA₂- and Myc₆-HA₂-Pex19p variants. (B) Direct interaction of Pex19p N-terminal region with Pex3p. ³⁵S-labeled, HA₂- and Myc₆-HA₂-tagged Pex19p mutants and [³⁵S]FL-Pex3p-EGFP were separately synthesized in a cell-free system. [³⁵S]HA₂-Pex19p and [³⁵S]Myc₆-HA₂-Pex19p variants (2.5 µl each) were incubated with [³⁵S]FL-Pex3p-EGFP (2.5 µl) and immunoprecipitated with anti-HA antibody. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Lanes 1-10, 10% input used for immunoprecipitation; lanes 11-20, immunoprecipitates. Solid arrowhead indicates [³⁵S]FL-Pex3p-EGFP and open arrowheads indicate [³⁵S]HA₂-Pex19p and [³⁵S]Myc₆-HA₂-Pex19p mutants.

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