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

被引:55
作者
Austin, C [1 ]
Hinners, I [1 ]
Tooze, SA [1 ]
机构
[1] Imperial Canc Res Fund, Secretory Pathway Lab, London WC2A 3PX, England
关键词
D O I
10.1074/jbc.M908875199
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:21862 / 21869
页数:8
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