Sar1 GTPase Activity Is Regulated by Membrane Curvature

Abstract

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5′-[(β,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.

SAR1 paralogs differ biochemically in assembly of the COPII coat

“…Enhanced recruitment of SEC23 may be due to the formation of SAR1 dimers on the membrane (25)(26)(27). In fact, we found a significant amount of soluble SAR1 recruited to the membrane, although we were unable to distinguish soluble SAR1B from SAR1A as the two have the same SDS-PAGE mobility.…”

“…It is easy to think of SAR1 as just a small part of a bigger COPII machinery, rather than focusing on the important roles SAR1 itself plays. Hannah et al (26) showed that the amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.…”

Membrane Transport at an Organelle Interface in the Early Secretory Pathway: Take Your Coat Off and Stay a While

“…The first is based on the premise that Sar1 action is restricted to the ER, where it promotes membrane tubulation and ultimately accumulates at the neck of nascent COPII‐coated buds to facilitate bilayer scission upon GTP hydrolysis. This idea is supported by studies demonstrating the ability of recombinant Sar1 to create membrane tubules (Figure ), as well as more recent work demonstrating that Sar1 is capable of sensing membrane curvature . Additionally, the intrinsic GTPase activity of Sar1 is stimulated in the presence of elevated curvature, suggesting a mechanism by which it may drive transport carrier scission .…”

“…This idea is supported by studies demonstrating the ability of recombinant Sar1 to create membrane tubules (Figure ), as well as more recent work demonstrating that Sar1 is capable of sensing membrane curvature . Additionally, the intrinsic GTPase activity of Sar1 is stimulated in the presence of elevated curvature, suggesting a mechanism by which it may drive transport carrier scission . In a second model, Sar1 not only acts at the ER, but also on free transport carriers en route to the ERGIC.…”

“…However, membrane penetration by the amino‐terminal amphipathic helix of Sar1 is critical to induce positive curvature. Specifically, when bound to GTP, Sar1 stably penetrates the lipid bilayer to a depth of ≈5 Å, just below the phospholipid headgroups of the outer leaflet . This type of insertion has been demonstrated to be conducive to outward membrane bending, which is likely stabilized by the sequential recruitment of Sec23‐Sec24 heterodimers that bind directly to activated Sar1 and Sec13‐Sec31 heterotetramers that associate with the inner coat .…”