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

The 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 atomic level knowledge of Arf1 activation by Sec7 domain-containing GEFs, our understanding of the biophysical processes regulating Arf1 and GBF1 dynamics is limited. We used Fluorescence Recovery After Photobleaching data and kinetic Monte Carlo simulation to assess behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses suggest Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1. Furthermore, the GBF1-mediated Arf1 activation is much faster than GBF1 cycling on/off the membrane, suggesting GBF1 is regulated by processes other than its interactions Arf1. Interestingly, modeling the behavior of the catalytically inactive GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1, and suggests GBF1/E794K is stabilized on the membrane independently of complex formation.

The structure of COPI vesicles and regulation of vesicle turnover

“…3A) [45,81,82,92–94]. These structures help explain the observed mutual stabilization of interaction with the membrane that occurs for GBF1 and Arf1 [87,88]. Subsequent binding of GTP to Arf1 leads to dissociation from the Sec7 domain.…”

“…Membrane association of GBF1 and Arf1 is essential for GBF1 to successfully displace GDP from Arf1 [86]. GBF1 and Arf1 can each form transient associations with the membrane, but the interaction between the two proteins mutually stabilizes the association of both proteins with the membrane [87]. After nucleotide exchange, Arf1 remains membrane bound while GBF1 dissociates from the membrane.…”

Critical Determinants in ER-Golgi Trafficking of Enzymes Involved in Glycosylation

“…The activation of the GTPase ADP-ribosylation factor 1 (Arf1) is a prerequisite for the assembly of the COPI coat [ 37 , 38 , 39 ]. Guanine nucleotide-exchange factors (GEFs) stimulate the GTPase Arf1 activation by exchanging GDP to GTP; then, the activated GTPase Arf1 embeds into the lipid membrane by using a myristoylated α-helix [ 40 , 41 ]. In turn, GTPase Arf1 recruits the coatomer complex with an inner coat and outer coat to transport protein and lipid cargo from the Golgi to the ER and between the Golgi cisternae [ 42 , 43 ].…”

Site-specific phosphorylations of the Arf activator GBF1 differentially regulate GBF1 function in Golgi homeostasis and secretion versus cytokinesis

“…One of the key regulators of the secretory pathway are the small GTPases of the Arf superfamily that facilitate traffic at the ER-Golgi interface 1 – 5 . Arfs are essential to recruit the heptameric coatomer coat complexes to Golgi membranes to form COPI vesicles that recycle proteins and membrane from the Golgi to the ER 6 – 9 . All Arfs cycle between GDP- and GTP-bound states and are functional and membrane-associated only when in their GTP-bound (active) state 10 .…”