THE ROLE OF ADP-RIBOSYLATION FACTOR AND PHOSPHOLIPASE-D IN ADAPTER RECRUITMENT

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). W e have used subcellular fractionation followed by Western blotting, as well, as immunofluorescence and immunogold electron microscopy, to in vestigate both the recruitment of AP-2 adaptors onto the plasma membra ne and their targeting to endosomes, and we have also examined the rec ruitment of AP-1 under the same conditions. Two lines of evidence indi cate that the GTP gamma S-induced targeting of AP-2 to endosomes is me diated by ADP-ribosylation factor-1 (ARF1). First, GTP gamma S loses i ts effect when added to ARF-depleted cytosol, but this effect is resto red 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, sugge sting that ARF may be acting catalytically. The ARFs have been shown t o activate phospholipase D (PLD), and we find that addition of exogeno us PLD has the same effect as GTP gamma S or Q71L ARF1. Neomycin, whic h inhibits endogenous PLD by binding to its cofactor phosphatidylinosi tol 4,5-bisphosphate, prevents the recruitment of AP-2 not only onto e ndosomes but also onto the plasma membrane, suggesting that both event s 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.