ARF6 TARGETS RECYCLING VESICLES TO THE PLASMA-MEMBRANE - INSIGHTS FROM AN ULTRASTRUCTURAL INVESTIGATION

We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPa se localizes to the plasma membrane and intracellular endosomal compar tments, Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another stu dy on the distribution of ARF6 in subcellular fractions of Chinese ham ster ovary (CHO) cells suggested that ARF6 did not localize to endosom es labeled after 10 min of horseradish peroxidase (HRP) uptake, but in stead was uniquely localized to the plasma membrane, and that its repo rted endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein ex pression by cryoimmunogold electron microscopy, ARF6 localized predomi nantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localiz e to early endocytic structures that labeled with HRP shortly after up take, Furthermore, we have shown that the ARF6-containing intracellula r compartment partially colocalized with transferrin receptors and cel lubrevin and morphologically resembled the recycling endocytic compart ment previously described in CHO cells. HRP labeling in cells expressi ng ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small per ipheral vesicles, whereas the mutant protein was enriched on plasma me mbrane invaginations. On the other hand, expression of ARF6(T27N), a m utant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HR P on prolonged exposure to the tracer. Taken together, our findings su ggest that ARF activation is required for the targeted delivery of ARF 6-positive, recycling endosomal vesicles to the plasma membrane.