Functional characterization of class I Arfs and their guanine nucleotide exchange factors at the Golgi complex.

摘要

We examined the function of ADP-ribosylation factors (Arfs) and their guanine nucleotide exchange factors (GEFs) that regulate recruitment of coat proteins on the Golgi complex. The large ArfGEF GBF1 localizes at the cis-Golgi complex while BIG1 and BIG2 localize at the trans-Golgi network (TGN). Complementary overexpression and RNA-based knockdown approaches established that GBF1 but not BIGs, is required for COPI recruitment, Golgi stack maintenance and sub-compartmentalization while BIGs appear specialized for clathrin adaptor recruitment and for assembly and maintenance of the TGN. Our observations disprove two widely accepted mechanisms for cargo export by establishing that COPII is the only coat required for sorting and export from the ER exit sites and that BIGs are not required for traffic of the cargo protein VSVG to the cell surface. Furthermore, we provide evidence that may ultimately explain how these ArfGEFs regulate different coats in spite of their well-characterized promiscuity towards class I and II Arfs. We prove for the first time that Arf3 is activated uniquely by BIGs at the TGN. Also, contrary to expectations, we demonstrate that Arf3 differs from Arf1 in regard to localization pattern as well as temperature sensitivity of membrane recruitment. Shifting temperature to 20°C for 2 hours, a method known to block cargo in trans-Golgi compartments, caused a dramatic redistribution Arf3 but not Arf1. Redistribution of Arf3 from Golgi membranes upon shift to 20°C was not immediate but occurred gradually over 20 minutes. Arf1 and Arf3 differ in sequence only in two short regions at the N- and C-termini. Analysis of swap constructs established that two amino acids in the N-terminal region of Arf3 and Arf1 are responsible for directing the temperature sensitivity while two amino acids in the C-terminus directs Arf3's specific localization. Arf3 knockdown had no impact on any of the markers tested or on VSVG trafficking to the cell surface. My work provides solid evidence to support that ArfGEFs function at different compartments to regulate membrane recruitment of specific coat proteins, and may also regulate distinct sets of Arfs that localize preferentially to these particular compartments.