Src activates retrograde membrane traffic through phosphorylation of GBF1

The Src tyrosine kinase controls cancer-critical protein glycosylation through Golgi to ER relocation of GALNTs enzymes. How Src induces this trafficking event is unknown. Golgi to ER transport depends on the GTP Exchange factor (GEF) GBF1 and small GTPase Arf1. Here we show that Src induces the formation of tubular transport carriers containing GALNTs. The kinase phosphorylates GBF1 on 10 tyrosine residues; two of them, Y876 and Y898 are located near the C-terminus of the Sec7 GEF domain. Their phosphorylation promotes GBF1 binding to the GTPase; molecular modeling suggests partial melting of the Sec7 domain and intramolecular rearrangement. GBF1 mutants defective for these rearrangements prevent binding, carrier formation and GALNTs relocation, while phosphomimetic GBF1 mutants induce tubules. In sum, Src promotes GALNTs relocation by promoting GBF1 binding to Arf1. Based on residue conservation, similar regulation of GEF-Arf complexes by tyrosine phosphorylation could be a conserved and wide-spread mechanism.

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

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.

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

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.

Src activates retrograde membrane traffic through phosphorylation of GBF1

The Src tyrosine kinase controls cancer-critical protein glycosylation through Golgi to ER relocation of GALNTs enzymes. How Src induces this trafficking event is unknown. Golgi to ER transport depends on the GTP Exchange factor (GEF) GBF1 and small GTPase Arf1. Here we show that Src induces the formation of tubular transport carriers containing GALNTs through the activation of a GBF1-Arf1 complex. The complex is initiated by phosphorylation on GBF1 on 10 tyrosine residues; two of them, Y876 and Y898 are located near the C-terminus of the Sec7 GEF domain. Their phosphorylation promotes partial melting of the Sec7 domain, favoring binding to the GTPase. Perturbation of these rearrangements prevent GALNTs relocation. In sum, Src promotes GALNTs relocation by favoring binding of GBF1 to Arf1. Regulation of a GEF-Arf axis by tyrosine phosphorylation appears to be a highly conserved and wide-spread mechanism.

Promiscuity of the catalytic Sec7 domain within the guanine nucleotide exchange factor GBF1 in ARF activation, Golgi homeostasis, and effector recruitment

The integrity of the Golgi and trans-Golgi network (TGN) is disrupted by brefeldin A (BFA), which inhibits the Golgi-localized BFA-sensitive factor (GBF1) and brefeldin A–inhibited guanine nucleotide-exchange factors (BIG1 and BIG2). Using a cellular replacement assay to assess GBF1 functionality without interference from the BIGs, we show that GBF1 alone maintains Golgi architecture; facilitates secretion; activates ADP-ribosylation factor (ARF)1, 3, 4, and 5; and recruits ARF effectors to Golgi membranes. Unexpectedly, GBF1 also supports TGN integrity and recruits numerous TGN-localized ARF effectors. The impact of the catalytic Sec7 domain (Sec7d) on GBF1 functionality was assessed by swapping it with the Sec7d from ARF nucleotide-binding site opener (ARNO)/cytohesin-2, a plasma membrane GEF reported to activate all ARFs. The resulting chimera (GBF1-ARNO-GBF1 [GARG]) targets like GBF1, supports Golgi/TGN architecture, and facilitates secretion. However, unlike GBF1, GARG activates all ARFs (including ARF6) at the Golgi/TGN and recruits additional ARF effectors to the Golgi/TGN. Our results have general implications: 1) GEF’s targeting is independent of Sec7d, but Sec7d influence the GEF substrate specificity and downstream effector events; 2) all ARFs have access to all membranes, but are restricted in their distribution by the localization of their activating GEFs; and 3) effector association with membranes requires the coincidental presence of activated ARFs and specific membrane identifiers.

RalF-Mediated Activation of Arf6 Controls Rickettsia typhi Invasion by Co-Opting Phosphoinositol Metabolism

Rickettsiae are obligate intracellular pathogens that induce their uptake into nonphagocytic cells; however, the events instigating this process are incompletely understood. Importantly, diverse Rickettsia species are predicted to utilize divergent mechanisms to colonize host cells, as nearly all adhesins and effectors involved in host cell entry are differentially encoded in diverse Rickettsia species. One particular effector, RalF, a Sec7 domain-containing protein that functions as a guanine nucleotide exchange factor of ADP-ribosylation factors (Arfs), is critical for Rickettsia typhi (typhus group rickettsiae) entry but pseudogenized or absent from spotted fever group rickettsiae. Secreted early during R. typhi infection, RalF localizes to the host plasma membrane and interacts with host ADP-ribosylation factor 6 (Arf6). Herein, we demonstrate that RalF activates Arf6, a process reliant on a conserved Glu within the RalF Sec7 domain. Furthermore, Arf6 is activated early during infection, with GTP-bound Arf6 localized to the R. typhi entry foci. The regulation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which generates PI(4,5)P 2 , by activated Arf6 is instrumental for bacterial entry, corresponding to the requirement of PI(4,5)P 2 for R. typhi entry. PI(3,4,5)P 3 is then synthesized at the entry foci, followed by the accumulation of PI(3)P on the short-lived vacuole. Inhibition of phosphoinositide 3-kinases, responsible for the synthesis of PI(3,4,5)P 3 and PI(3)P, negatively affects R. typhi infection. Collectively, these results identify RalF as the first bacterial effector to directly activate Arf6, a process that initiates alterations in phosphoinositol metabolism critical for a lineage-specific Rickettsia entry mechanism.