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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
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 GTPS-induced targeting of
AP-2 to endosomes is mediated by ADP-ribosylation factor-1 (ARF1). First, GTPS 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
GTPS. 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 GTPS 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.
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