[31] Functional analysis of ADP-ribosylation factor (ARF) guanine nucleotide exchange factors Gea1p and Gea2p in yeast

This chapter presents the functional analysis of adenosine diphosphate (ADP)-ribosylation factor (ARF) guanine nucleotide exchange factors Gea1p and Gea2p in yeast. The yeast Gealp protein has not been purified to homogeneity but useful information has been obtained as to its function from partial purification of a His6-tagged version of the protein from yeast. The chapter also describes purification of myristoylated yeast ARF2 from Escherichia coli and an assay for measuring exchange of guanosine triphophate (GTP) for guanosine diphosphate (GDP) on myristoylated yeast ARF2. The yeast ARF1 and ARF2 proteins (which share approximately the same level of sequence homology to both class I and class II mammalian ARFs) are 96% identical and are functionally interchangeable. Coelution of His6-Gealp and Scp160p from the Ni2+ column is described. One-tenth the volume of fractions is loaded onto a 6% sodium dodecyl sulphate -polyacrylamide gel and analyzed by Western blot using polyclonal antisera against Scpl60p and Gealp. ARF is purified in its GDP-bound form, and hence exchange activity can be assayed by incubating ARF-GDP with [35S]GTPγS and determining the rate of accumulation of [35S]GTPγS-ARF.