Direct and GTP-dependent interaction of ADP-ribosylation factor 1 with clathrin adaptor protein AP-1 on immature secretory granules

ADP-ribosylation factor 1 (ARF1) mediates clathrin coat formation on PC12 i mmature secretory granules (ISGs). We have used two approaches to investiga te whether ARF1 interacts directly with the clathrin adaptor protein, AP-1, Using an in vitro recruitment assay and co-immunoprecipitation, we could i solate an AP-1 .ARF1 complex. Then we used a site-directed photocross-linki ng approach to determine the components that act downstream of ARF1 in clat hrin coat formation on ISGs. Myristoylated ARF1, with a photolabile phenyla lanine analogue incorporated into its putative effector domain (switch 1), showed a specific, GTP dependent interaction with both the gamma- and beta- adaptin subunits of AP-1 on ISGs. These experiments provide evidence for a direct interaction of ARF1 with AP-1. On mature secretory granules myristoy lated ARF1 does not bind, and hence clathrin coat formation cannot be initi ated, supporting the hypothesis that molecules involved in coat recruitment are removed during ISG maturation.