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© The Rockefeller University Press, 0021-9525/1998//603 $5.00
The Journal of Cell Biology, Volume 140, Number 3, , 1998 603-616

Article

ARF6 Targets Recycling Vesicles to the Plasma Membrane: Insights from an Ultrastructural Investigation



Crislyn D'Souza-Schorey*, Elly van Donselaar{ddagger}, Victor W. Hsu§, Chunzhi Yang*, Philip D. Stahl*, and Peter J. Peters{ddagger}

* Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110; {ddagger} Department of Cell Biology and the Graduate School of Biomembranes, University of Utrecht Medical School, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands; and § Department of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

We have shown previously that the ADP- ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly 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 localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

Abbreviations used in this paper: ARF, ADP-ribosylation factor; Tfn-R, transferrin receptor; HA, hemagglutinin; MPR, cation-dependent mannose-6-phosphate receptor; LAMP, lysosome-associated membrane protein; PLD, phospholipase D; PIP2, phosphatidylinositol 4,5, bisphosphate; SNARE, soluble NSF attachment protein (SNAP) receptor; VAMP, vesicle-associated membrane protein.

We sincerely thank Hans Geuze and Willem Stoorvogel for critical comments on the manuscript and for insightful and helpful discussions during these studies. We also thank, Judith Klumpermann, Viola Oorschot, and Janice Griffiths for helpful suggestions with labeling procedures; Elizabeth Wolffe for help with generation of recombinant vaccinia virus; Guangpu Li for helpful discussions; Jeanette Leusen for reading the manuscript; and Tom van Rijn, George Posthuma, Mauritz Niekerk, and Rene Schriwanek for excellent technical assistance with photography and handling of EM micrographs.


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