The GAP Domain and the SNARE, Coatomer and Cargo Interaction Region of the ArfGAP2/3 Glo3 are Sufficient for Glo3 Function

Abstract

† These authors contributed equally to this work.The ArfGAP Glo3 is required for coat protein I vesicle generation in the Golgi-endoplasmic reticulum (ER) shuttle. The best-understood role of Glo3 is the stimulation of the GTPase activity of Arf1. In this study, we characterized functional domains of the ArfGAP Glo3 and identified an interaction interface for coatomer, SNAREs and cargo in the central region of Glo3 (BoCCS region). The GAP domain together with the BoCCS region is necessary and sufficient for all vital Glo3 functions. Expression of a truncated Glo3 lacking the GAP domain results in a dominant negative growth phenotype in glo3 cells at 37• C. This phenotype was alleviated by mutating either the BoCCS region or the Glo3 regulatory motif (GRM), or by overexpression of ER-Golgi SNAREs or the ArfGAP Gcs1. The GRM is not essential for Glo3 function; it may act as an intrinsic sensor coupling GAP activity to SNARE binding to avoid dead-end complex formation at the Golgi membrane. Our data suggest that membrane-interaction modules and cargo-sensing regions have evolved independently in ArfGAP1s versus ArfGAP2/3s.

Recruitment of Arf1-GDP to Golgi by Glo3p-Type ArfGAPs Is Crucial for Golgi Maintenance and Plant Growth

“…In animal cells and yeast, Glo3p-type ArfGAPs played roles in the assembly of COPI vesicles and also copurified with COPI vesicles (Lewis et al, 2004;Frigerio et al, 2007;Kliouchnikov et al, 2009;Saitoh et al, 2009;Schindler et al, 2009;Kartberg et al, 2010). Furthermore, in yeast cells, Glo3p at high levels can suppress the temperature-sensitive arf1-3 mutant, a GDP-bound dominant negative form (Zhang et al, 1998).…”

“…To obtain insight into the physiological roles of AGDs, we focused on one subfamily that contains three AGD isoforms, AGD8, AGD9, and AGD10. They exhibit the closest relationship to the Glo3p-type ArfGAPs that are involved in COPI vesicle biogenesis at the Golgi apparatus (Schindler et al, 2009;Spang et al, 2010). The public microarray data revealed that AGD8 and AGD9 are expressed at high levels in most tissues, whereas AGD10 is strongly expressed in stamens, moderately in root xylem cells, but virtually undetectable in other tissues (Supplemental Fig.…”

“…ArfGAP1 plays a role in increasing the affinity of coatomer for cargo, and the Glo3p-type ArfGAPs (Glo3p, ArfGAP2, and ArfGAP3) function in the biogenesis of COATOMER PROTEIN I (COPI) vesicles in animal cells and yeast (Lewis et al, 2004;Lee et al, 2005;Kliouchnikov et al, 2009;Saitoh et al, 2009;Schindler et al, 2009;Spang et al, 2010). A large number of ArfGAPs in both animal and plant cells have been identified and are classified into several subfamilies (Inoue and Randazzo, 2007).…”

Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

“…Further complicating the problem, is evidence that implicate ArfGAP proteins as positive regulators of the COPI coat rather than negative regulators: overexpression of any of the four yeast ArfGAPs suppressed the lethality of an arf1 mutant (Zhang et al 1998(Zhang et al , 2003. Further yeast experiments also support an active role for Gcs1 and Glo3 in cargo selection, acting on SNARE proteins prior to incorporation into vesicles to promote Arf1 and coatomer interaction (Rein et al 2002;Schindler and Spang 2007;Schindler et al 2009). Clearly, the precise role of the GAP in the COPI system remains to be fully understood, complicated by conflicting results from different labs and/or systems and may in fact be multifaceted by serving both positive and negative roles at different stages during the vesicle formation process (Spang et al 2010).…”

ArfGAP1 promotes COPI vesicle formation by facilitating coatomer polymerization

“…The function of ArfGAP1, 2 and 3 is, at least in part, related to interaction with the substrate protein Arf1•GTP, vesicle coat protein and the cargo proteins that are being transported. [8][9][10][11][12][13][14][15] The molecular basis for retrograde traffic between the Golgi cisternae or from the Golgi apparatus to the ER has been extensively studied. 2,[4][5][6][7]16 In these pathways, cargo is transferred from trans-side cisternae to the more cis-side, or from the Golgi apparatus to the endoplasmic reticulum (ER).…”