Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat

Abstract

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p-and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/ disassembly.

Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

“…Indeed, when the curvature-inducing amphipathic helix of Sar1 is replaced with an N-terminal histidine tag to drive recruitment to Ni-containing liposomes, subsequent recruitment of Sec23/Sec24 and Sec13/Sec31 is sufficient to drive the generation of spherical buds that remain attached to the donor liposome (Lee et al 2005). Thus an additional function of the Sar1 helix is to drive vesicle scission, a model supported by experiments that link GTPase activity to vesicle release in a manner analogous to that proposed for dynamin (Pucadyil and Schmid 2009;Kung et al 2012). Although the concave face of Sec23/Sec24 may also contribute to membrane curvature, it has been suggested that the relatively paltry dimer interface between these two molecules is not robust enough to impart curvature despite an intimate interaction with the lipid bilayer (Zimmerberg and Kozlov 2006).…”

“…One solution to this conundrum is that constant Sec12 GEF activity feeds new coat elements into a nascent bud (Futai et al 2004;Sato and Nakano 2005); coat release from the membrane might also be delayed by the increased affinity afforded by cargo proteins (Sato and Nakano 2005). However, recent findings suggest that a GAP inhibitory function, contributed by the peripheral ER protein, Sec16, also modulates the activity of the coat (Kung et al 2012;Yorimitsu and Sato 2012). Sec16 is a large, essential protein that associates with the cytoplasmic face of the ER membrane at ERES (Espenshade et al 1995;Connerly et al 2005).…”

“…It interacts with all of the COPII coat proteins (Gimeno et al 1996;Shaywitz et al 1997) and is thus thought to scaffold and/or organize coat assembly at these discrete domains (Supek et al 2002;Shindiapina and Barlowe 2010). In addition to this recruitment function, a fragment of Sec16 dampens the GAP-stimulatory effect of Sec31, probably by preventing Sec31 recruitment to Sar1/Sec23/Sec24 (Kung et al 2012). The GAP-inhibitory effect of Sec16 was diminished in the context of a point mutation in Sec24 (Kung et al 2012), raising the tantalizing possibility that cargo engagement by Sec24 could trigger interaction with Sec16 to inhibit the full GTPase activity of the coat in such a manner that a vesicle is initiated around a cargo-bound complex of Sar1/Sec23/Sec24/Sec16 (Springer et al 1999).…”

Apoptosis-linked Gene-2 (ALG-2)/Sec31 Interactions Regulate Endoplasmic Reticulum (ER)-to-Golgi Transport

“…The interactions may facilitate vesicle budding and cargo loading. Cargo loading is only beginning to be understood and appears to involve subtly timed interactions by multiple ERES components to properly regulate vesicle size and content (60,61). The functional locus of the ALG-2/Sec31A interaction could also be shortly after vesicle formation.…”

Les gènes et les protéines qui contrôlent la voie de sécrétion

“…Deux stagiaires post-doctoraux successifs, Joe Campbell et Frantisek Supek, ont défini les conditions dans lesquelles on pouvait observer un rôle de Sec16p dans la réaction de bourgeonnement (Campbell & Schekman, 1997 ;Supek et al, 2002). Eugene Futai a réussià purifier le Sec16p recombinant et réuni les conditions dans lesquelles il contrôlait le cycle de la GTPase, médié par l'interaction de toutes les protéines COPII et de Sar1 (Kung et al, 2011). Pourtant maintenant encore, il n'est pas clair que Sec16 participe activement au cycle de bourgeonnement des vésicules ou joue plutôt un rôle régulateur en organisant les protéines COPII au site de sortie du RE.…”