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

. 2000 Jul 21;275(29):21862-9.

doi: 10.1074/jbc.M908875199.

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

C Austin et al. J Biol Chem. 2000.

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Abstract

ADP-ribosylation factor 1 (ARF1) mediates clathrin coat formation on PC12 immature secretory granules (ISGs). We have used two approaches to investigate whether ARF1 interacts directly with the clathrin adaptor protein, AP-1. Using an in vitro recruitment assay and co-immunoprecipitation, we could isolate an AP-1.ARF1 complex. Then we used a site-directed photocross-linking approach to determine the components that act downstream of ARF1 in clathrin coat formation on ISGs. Myristoylated ARF1, with a photolabile phenylalanine 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 myristoylated ARF1 does not bind, and hence clathrin coat formation cannot be initiated, supporting the hypothesis that molecules involved in coat recruitment are removed during ISG maturation.

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