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Updated: Aug 4, 2026

In vivo and in vitro Studies of Adaptor-clathrin Interaction
Published on: January 27, 2011
Direct and GTP-dependent interaction of ADP-ribosylation factor 1 with clathrin adaptor protein AP-1 on immature
C Austin1, I Hinners, S A Tooze
1Secretory Pathway Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX, United Kingdom.
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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