<p>HeLa cells expressing the indicated rab4 and rab5 constructs (green) were labeled with a monoclonal antibody against δ-adaptin (red). Rab4 localized with AP-3 predominantly on more peripheral endosomes, while rab5 did not (insets) (<b>A</b>). The extent of overlap between rab4 or rab4 mutants and AP-3 was quantified and showed that it was dependent on the GTP-bound form of rab4 (<b>B</b>). HeLa cells were co-transfected with VSVG-rabip4’ and the indicated rab4 constructs, and labeled for rabip4’ with a rabbit antibody (red) and δ-adaptin with a mouse antibody (blue). Rab4, rabip4’, and AP-3 colocalized in the perinuclear area independent of the nucleotide status of rab4 (insets, arrows). Active rab4 and AP-3 colocalized on endosomes closer to the cell periphery (arrowheads). Scale bar is 10 µm (<b>C</b>). The degree of colocalization between AP-3 and rabip4’ in the absence (control) and in the presence of rab4 or rab4 mutants was quantified. Expression of rab4N121I induced a 2-fold increase in colocalization between rabip4’ and AP-3. Scale bar is 10 µm (<b>D</b>). Ultrathin cryosections of HeLa cells transfected with VSVG-rabip4’ and rab4S22N were triple-immunogold labeled for AP-3 (15 nm gold), VSVG (10 nm gold), and rab4 (5 nm gold, indicated by arrows). AP-3 and rabip4’ colocalize on typical recycling tubules (arrow) in the vicinity of endosomal vacuoles (E). Bars, 200 nm (<b>E</b>).</p
