Modeling the dynamic behaviors of the COPI vesicle formation regulators, the small GTPase Arf1 and its activating Sec7 guanine nucleotide exchange factor GBF1 on Golgi membranes

Abstract

ABSTRACTThe components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain-containing guanine nucleotide exchange factor GBF1. This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat. Although we have an atomic level knowledge of Arf1 activation by Sec7 domain-containing GEFs, our understanding of the biophysical parameters that regulate Arf1 and GBF1 association with Golgi membranes and with each other is limited. We used Fluorescence Recovery After Photobleaching (FRAP) data and kinetic Monte Carlo simulation based on continuous-time random walk to assess behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses support a model in which Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1, and in which Arf1 activation is much faster than GBF1 cycling on the membrane. Interestingly, modeling the behavior of the GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1, and suggests that its prolonged association with the membrane occurs independently of complex formation.