Inp1p binds directly to the cytosolic domain of Pex3p in vitro. GST-Pex3p (40–441) and GST were bound to glutathione Sepharose beads and incubated with a detergent lysate of spheroplasts expressing HA-tagged Inp1p and Mvp1p at endogenous levels (A). After extensive washing, the bound fraction and lysate were analyzed by SDS-PAGE and immunoblotting using the HA monoclonal 12CA5. Yeast lysates (YL) represent 5% of the lysate added to the beads and analyzed by blotting. Because the signal of Inp1p-HA was too low in the YL, 5 times more lysate was reloaded on a separate gel and compared with the GST- and GST-Pex3–bound fraction (right-hand panel). Bottom panel shows Coomassie staining. (B) GST-Inp1p and GST were bound to glutathione Sepharose and incubated with a lysate of E. coli expressing either 6xHIS-tagged Pex3p (40–441) or HIS-tag only, or with lysis buffer only (−). After extensive washing, bound fractions were analyzed by SDS-PAGE and Coomassie staining of the gel. A lane was included with partially purified 6xHIS-Pex3p as control. M, molecular weight marker. Arrow indicates 6xHIS-Pex3p. Asterisks indicate multiple GST-Inp1p fragments. EL, E. coli lysate of 6xHIS-Pex3p–expressing cells.