Characterization of a Fast Cycling ADP-ribosylation Factor 6 Mutant

Lorraine C Santy

doi:10.1074/jbc.c200481200

Abstract

Studies of GTPase function often employ expression of dominant negative or constitutively active mutants. Dominant negative mutants cannot bind GTP and thus cannot be activated. Constitutively active mutants cannot hydrolyze GTP and therefore accumulate a large pool of GTP-bound GTPase. These mutations block the normal cycle of GTP binding, hydrolysis, and release. Therefore, although the GTPase-deficient mutants are in the active conformation, they do not fully imitate all the actions of the GTPase. This is particularly true for the ADP-ribosylation factors (ARFs), GTPases that regulate vesicular trafficking events. In Ras and Rho GTPases replacement of phenylalanine 28 with a leucine residue produces a "fast cycling" mutant that can undergo spontaneous GTP-GDP exchange and retains the ability to hydrolyze GTP. Unfortunately this phenylalanine residue is not conserved in the ARF family of GTPases. Here we report the design and characterization of a novel activated mutant of ARF6, ARF6 T157A. In vitro studies show that ARF6 T157A can spontaneously bind and release GTP more quickly than the wild-type protein suggesting that it is a fast cycling mutant. This mutant has enhanced activity in vivo and induces cortical actin rearrangements in HeLa cells and enhanced motility in Madin-Darby canine kidney cells.

Highlights

Studies of GTPase function often employ expression of dominant negative or constitutively active mutants
If ARF6 T157A truly is a fast cycling mutant, it should have enhanced rates of GTP binding and release when compared with the wild-type protein
The loading of ARF6 T157A with GTP was investigated in vitro to determine whether this mutation enhances the spontaneous rate of GTP binding

Summary

Introduction

Studies of GTPase function often employ expression of dominant negative or constitutively active mutants. Active mutants cannot hydrolyze GTP and accumulate a large pool of GTP-bound GTPase These mutations block the normal cycle of GTP binding, hydrolysis, and release. The GTPase-deficient mutants are in the active conformation, they do not fully imitate all the actions of the GTPase This is true for the ADP-ribosylation factors (ARFs), GTPases that regulate vesicular trafficking events. Active mutants, on the other hand, cannot hydrolyze the bound GTP leading the cell to accumulate a large pool of activated GTPase Both of these mutations block the normal cycle of GTP binding followed by hydrolysis. Constitutively active mutants cannot necessarily recapitulate all of the actions of the normal GTPase This is true of ARF-regulated trafficking events, such as budding of COPI-coated vesicles from the Golgi. ARF6 Q67L alters cell morphology and has toxic effects with extended expression [13, 15, 16]

Methods

Results

Conclusion