Activation of toxin ADP-ribosyltransferases by the family of ADP-ribosylation factors.

Abstract

ADP-ribosylation factors or ARFs are 20-kDa guanine nucleotide-binding proteins, initially identified as stimulators of cholera toxin-catalyzed ADP-ribosylation of Gs alpha. We now know that ARFs play a critical role in many vesicular trafficking events and ARF activation of a membrane-associated phospholipase D (PLD) has been recognized. ARF is active and associates with membranes when GTP is bound. The active state is terminated by hydrolysis of bound GTP, producing inactive ARF-GDP. The nucleotide effect on ARF association with membranes is related to alteration in orientation of the N-terminal myristoyl moiety that is important for ARF function. Cycling of ARF between active and inactive states involves guanine nucleotide-exchange proteins (GEPs) that accelerate replacement of bound GDP with GTP and GTPase-activating proteins (GAPS) that are responsible for ARF inactivation. Six mammalian ARFs have been identified by cDNA cloning. Class I ARFs 1 and 3 have been studied most extensively. Their activation (GTP binding) is catalyzed by a GEP now purified from spleen cytosol. In crude preparations, GEP was inhibited by brefeldin A (BFA), which in cells causes apparent disintegration of Golgi. Demonstration that the approximately 60 kDa purified GEP was not inhibited by BFA means that contrary to earlier belief, there must be another protein to mediate BFA inhibition. GEP activity was greatly enhanced by phosphatidyl serine. The purified GEP, equally active with ARFs 1 and 3, was inactive with ARFs 5 and 6 (Classes II and III); myristoylated ARFs were better substrates than were their non-myristoylated counterparts. ARF GAP purified from bovine spleen cytosol in our laboratory had much broader substrate specificity than the GEP. It used both ARFs 5 and 6 at least as well as ARFs 1 and 3; myristoylation was without effect. It also accelerated GTP hydrolysis by certain ARF mutants and an ARF-like protein (ARL1) that does not have ARF activity. The purified GAP also differed from the GEP in its rather specific requirement for phosphatidylinositol bisphosphate. This was also observed with a seemingly different ARF GAP that was purified and subsequently cloned in Cassel's laboratory. Activation and inactivation of ARFs present many potential sites for physiological regulation and, therefore, for pathological disruption of ARF function.