The Arf1p GTPase-activating protein Glo3p executes its regulatory function through a conserved repeat motif at its C-terminus

N Yahara,K Sato,A Nakano

doi:10.1242/jcs.02997

Abstract

ADP-ribosylation factors (Arfs), key regulators of intracellular membrane traffic, are known to exert multiple roles in vesicular transport. We previously isolated eight temperature-sensitive (ts) mutants of the yeast ARF1 gene, which showed allele-specific defects in protein transport, and classified them into three groups of intragenic complementation. In this study, we show that the overexpression of Glo3p, one of the GTPase-activating proteins of Arf1p (ArfGAP), suppresses the ts growth of a particular group of the arf1 mutants (arf1-16 and arf1-17). Other ArfGAPs do not show such a suppression activity. All these ArfGAPs show sequence similarity in the ArfGAP catalytic domain, but are divergent in the rest of molecules. By domain swapping analysis of Glo3p and another ArfGAP, Gcs1p, we have shown that the non-catalytic C-terminal region of Glo3p is required for the suppression of the growth defect in the arf1 ts mutants. Interestingly, Glo3p and its homologues from other eukaryotes harbor a well-conserved repeated ISSxxxFG sequence near the C-terminus, which is not found in Gcs1p and its homologues. We name this region the Glo3 motif and present evidence that the motif is required for the function of Glo3p in vivo.

Highlights

ADP-ribosylation factors (Arfs) constitute a ubiquitous family of small GTPases in eukaryotes, and are recognized as essential components in membrane traffic
ArfGAP, Gcs1p, we have shown that the non-catalytic Cterminal region of Glo3p is required for the suppression of the growth defect in the arf1 ts mutants
GLO3 is a multicopy suppressor of arf1-16 and arf1-17 To identify genes that participate in multiple functions of Arf1p, we screened the S. cerevisiae genomic DNA library on a multicopy vector for plasmids that suppress the growth defect of arf1-16 at 35°C

Summary

Introduction

ADP-ribosylation factors (Arfs) constitute a ubiquitous family of small GTPases in eukaryotes, and are recognized as essential components in membrane traffic. There are six Arf proteins in mammals and three Arfs in the yeast Saccharomyces cerevisiae, which are classified into three and two classes, respectively These Arf proteins regulate formation of vesicles in a variety of steps of membrane traffic by interacting with coat components and appear to control phospholipid metabolism and cytoskeletal organization in various ways (Donaldson and Jackson, 2000; Kirchhausen, 2000; Nie et al, 2003; Schekman and Orci, 1996). Among the three Arf proteins, Arf1p and Arf2p are 96% identical in amino acid sequences They execute redundant essential functions for growth and their double knockout is lethal (Stearns et al, 1990), whereas Arf3p is dispensable

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