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© The Rockefeller University Press, 0021-9525/1997//1239 $5.00
The Journal of Cell Biology, Volume 138, Number 6, , 1997 1239-1254

Article

The Role of ADP-ribosylation Factor and Phospholipase D in Adaptor Recruitment



Michele A. West, Nicholas A. Bright, and Margaret S. Robinson

University of Cambridge, Department of Clinical Biochemistry, Cambridge CB2 2QR, United Kingdom

AP-1 and AP-2 adaptors are recruited onto the TGN and plasma membrane, respectively. GTP{gamma}S stimulates the recruitment of AP-1 onto the TGN but causes AP-2 to bind to an endosomal compartment (Seaman, M.N.J., C.L. Ball, and M.S. Robinson. 1993. J. Cell Biol. 123:1093–1105). We have used subcellular fractionation followed by Western blotting, as well as immunofluorescence and immunogold electron microscopy, to investigate both the recruitment of AP-2 adaptors onto the plasma membrane and their targeting to endosomes, and we have also examined the recruitment of AP-1 under the same conditions. Two lines of evidence indicate that the GTP{gamma}S-induced targeting of AP-2 to endosomes is mediated by ADP-ribosylation factor-1 (ARF1). First, GTP{gamma}S loses its effect when added to ARF-depleted cytosol, but this effect is restored by the addition of recombinant myristoylated ARF1. Second, adding constitutively active Q71L ARF1 to the cytosol has the same effect as adding GTP{gamma}S. The endosomal membranes that recruit AP-2 adaptors have little ARF1 or any of the other ARFs associated with them, suggesting that ARF may be acting catalytically. The ARFs have been shown to activate phospholipase D (PLD), and we find that addition of exogenous PLD has the same effect as GTP{gamma}S or Q71L ARF1. Neomycin, which inhibits endogenous PLD by binding to its cofactor phosphatidylinositol 4,5-bisphosphate, prevents the recruitment of AP-2 not only onto endosomes but also onto the plasma membrane, suggesting that both events are mediated by PLD. Surprisingly, however, neither PLD nor neomycin has any effect on the recruitment of AP-1 adaptors onto the TGN, even though AP-1 recruitment is ARF mediated. These results indicate that different mechanisms are used for the recruitment of AP-1 and AP-2.

Abbreviations used in this paper: AFR, ADP-ribosylation factor; BFA, brefeldin A; NRK, normal rat kidney; PA, phosphatidic acid; PIP2, phosphatidylcholine 4,5-bisphosphate; PLD, phospholipase D.

Address all correspondence to Margaret S. Robinson, University of Cambridge, Department of Biochemistry, Cambridge CB2 2QR, U.K. Tel.: 44 1223 330163. Fax: 44 1223 330598.

Michele A. West's current address is Department of Biochemistry, University of Dundee, Dundee DD1 4HN, Scotland.

We are greatly indebted to Nick Ktistakis for helping us with the PLD assay. We also thank Sally Gray for assistance in the construction of the ARF fusion proteins; Howard Davidson, Bill Balch (The Scripps Research Institute, La Jolla, CA) and Sharon Tooze (ICRF, London, UK) for plasmids; Paul Luzio for the anti-lgp110 antiserum; Nick Ktistakis and Mike Roth (University of Texas Southwestern Medical Center, Dallas, TX) for communicating unpublished results; and Rainer Duden, John Kilmartin, Nick Ktistakis, Paul Luzio, and members of the Robinson lab for reading the manuscript and for helpful discussions.


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