Rabip4’ interacts specifically and directly with AP-3.

Abstract

<p>Immobilized GST-rabip4’ (aa 299–708) was incubated with brain cytosol. Bound proteins were resolved by SDS-PAGE and analyzed by tandem mass spectrometry, which yielded β3-adaptin as binding partner (<b>A</b>). Eluates were probed with antibodies against subunits of adaptor complexes showing specificity for AP-3 (<b>B</b>). GST-rabip4’ beads were incubated with detergent extracts from rescued <i>mocha</i> cells and bound proteins were analyzed by Western blot for the indicated AP-3 and AP-1 subunits. The ubiquitous AP-3 specifically interacted with rabip4’ (<b>C</b>). AP-3 was immunoprecipitated from lysates of HeLa cells expressing VSVG-rabip4’ and analyzed by Western blot with antibodies against VSVG and AP-3 subunits. Immunoprecipitation of AP-1 or a monoclonal antibody against HA (control IgG) did not co-immunoprecipitate VSVG-rabip4’ (<b>D</b>). GST-rabip4’ was immobilized on GSH beads and incubated with ³⁵S-labeled AP-3 subunits. Rabip4’ interacted directly with AP-3 through the β3 subunit (<b>E</b>).</p