29] Expression, purification, and biochemical properties of EFA6, a Sec7 domain-containing guanine exchange factor for ADP-ribosylation factor 6 (ARF6)

Abstract

This chapter presents method to produce and analyze the biochemical activity of EFA6 as an ARF6- specific guanine nucleotide exchange factors (GEF) and to assess its intracellular localization in mammalian cells. For analysis of bound nucleotides on recombinant ARF6, recombinant ARF1 is purified as a GDP-bound form and tryptophan fluorescence is used to determine the nature of the nucleotide bound to purify ARF6. Activation of ARF1 by GEFs occurs only when the ARF/GEF complex is associated with membranes. In the case of ARF1, the myristoyl moiety that increases membrane association of both the GDP- and GTP-bound forms is required for catalyzed nucleotide exchange. Therefore, myristoylated ARF6 is produced with the aim of analyzing the nucleotide exchange activity of EFA6. To assess the extent of myristoylation, modified ARF6 is discriminated from the unmodified form by its increased affinity for membrane phospholipids. In addition, the ability of recombinant EFA6 to catalyze the binding of guanosine 5'-[γ-thio]triphosphate ([ 35 S]GTPγS) is monitored on myrARF6.