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 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.
Austin
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Secretory Pathway Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX, United Kingdom.
Hinners
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Tooze
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D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D002966:Clathrin / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D008565:Membrane Proteins; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1