Regulation of GTP Hydrolysis on ADP-ribosylation Factor-1 at the Golgi Membrane

Abstract

The interaction of the coatomer coat complex with the Golgi membrane is initiated by the active, GTP-bound state of the small GTPase ADP-ribosylation factor 1 (ARF1), whereas GTP hydrolysis triggers coatomer dissociation. The hydrolysis of GTP on ARF1 depends on the action of members of a family of ARF1-directed GTPase-activating proteins (GAPs). Previous studies in well defined systems indicated that the activity of a mammalian Golgi membrane-localized ARF GAP (GAP1) might be subjected to regulation by membrane lipids as well as by the coatomer complex. Coatomer was found to strongly stimulate GAP-dependent GTP hydrolysis on a membrane-independent mutant of ARF1, whereas we reported that GTP hydrolysis on wild type, myristoylated ARF1 loaded with GTP in the presence of phospholipid vesicles was coatomer-independent. To investigate the regulation of ARF1 GAPs under more physiological conditions, we studied GTP hydrolysis on Golgi membrane-associated ARF1. The activities at the Golgi of recombinant GAP1 as well as coatomer-depleted fractions from rat brain cytosol resembled those observed in the presence of liposomes; however, unlike in liposomes, GAP activities on Golgi membranes were approximately doubled upon addition of coatomer. By contrast, endogenous GAP activity in Golgi membrane preparations was unaffected by coatomer. Cytosolic GAP activity was partially reduced following immunodepletion of GAP1, indicating that GAP1 plays a significant although not exclusive role in the regulation of GTP hydrolysis at the Golgi. Unlike the activities of the mammalian proteins, the Saccharomyces cerevisiae Glo3 ARF GAP displayed activity at the Golgi that was highly dependent on coatomer. We conclude that ARF GAPs in themselves can efficiently stimulate GTP hydrolysis on ARF1 at the Golgi, and that coatomer may play an auxiliary role in this reaction, which would lead to an increased cycling rate of ARF1 in COPI-coated regions of the Golgi membrane.The budding of vesicles mediating the transport of proteins among different compartments of the secretory system is driven by the attachment of specialized protein complexes termed coats to the cytoplasmic surface of the donor membrane. These coat complexes also function in the sorting of appropriate cargo into the transport vesicle (reviewed in Refs.

ARFGAP2 and ARFGAP3 Are Essential for COPI Coat Assembly on the Golgi Membrane of Living Cells

“…Of particular interest will be to study the GAP activity of ARFGAP2 and ARFGAP3 and how they are regulated by interactions with other proteins. Strikingly, in an in vitro assay for GAP activity on Golgi membranes, GTP hydrolysis, supplied by yeast Glo3p, was increased 50-fold by the addition of coatomer (59). This suggests that coatomer-dependent localization of the Glo3-type ARFGAPs to the Golgi could be important for catalytic activity as well.…”

Exo1: A new chemical inhibitor of the exocytic pathway

“…We consider it likely that Exo1 affects the balance of the futile cycle with the functional cycle of ARF1-GTP hydrolysis (see Appendix VII). ARF-GAP1 has two possible fates when it is recruited to Golgi membranes from the cytosol: either it associates with unloaded COPI coatomers and ARF1-GTP, in which case it is highly active and ARF1 has high GTPase activity (the futile cycle), or it associates with COPI coatomers that are bound to cargo receptors, in which case it gives lower activity and allows ARF1-GTP to persist for longer (functional cycle, Appendix VII) (25)(26)(27). The effects of Exo1 on GAP-dependent ARF1-GTP hydrolysis might result from interference with the futile cycle hydrolysis step, the functional cycle hydrolysis step, or both.…”

Golgi Localization Determinants in ArfGAP1 and in New Tissue-specific ArfGAP1 Isoforms

“…GAP Activity Assays-Measurement of GAP activity on Golgi membranes was carried out as previously described (18). Briefly, recombinant myristoylated Arf1 (32) was loaded with [␥-32 P]GTP onto rat liver Golgi membranes (33) using the guanine nucleotide exchange activity that is present in the membrane.…”

“…15 and 16). Evidence for the Golgi function of the mammalian proteins includes their localization at the Golgi complex (11,13,14), their functional (17,18) and physical (14,19,20) interaction with the COPI coat, and the perturbation of Golgi integrity upon overexpression of the ArfGAP1 protein (21).…”