Sar1p-GTP is a strong inducer of membrane curvature
Activated Sar1p induces highly curved membrane tubules with narrow diameters, down to ≈26 nm26. For a dynamic in situ view, we observed the tubulation of lipid-labeled GUVs, using fast line-scanning microscopy (Fig. 1). Incubations contained Sar1p, Sec12ΔCp1 and a GTP-regenerating system (GTPr) to maximize Sar1p loading with GTP. To avoid osmotic effects, osmolarities were adjusted. Rapid tubulation increased membrane tension, resulting in GUV rupture and collapse into dense balls of soft tubules (Fig. 1b–d, Movie S1). Because GTP hydrolysis by Sar1 is slow in the absence of GTPase activating proteins of the outer coat, wtSar1p with hydrolysable GTP (Fig. 1b–e), the mutant Sar1p(H77L) with GTP (Fig. S1d) and wtSar1p with non-hydrolysable GMP-PNP all resulted in liposome tubulation. In incubations of wtSar1p and Sar1p(H77L) with GTP, prominent straight and rigid tubules appeared (Fig. 1e and Fig. S1d,6). These exhibited brighter fluorescence and sometimes fraying ends, suggesting bundles. Control incubations without active Sar1p (Fig. 1a, S1a–c) yielded mostly intact GUVs with a low level of tubules and pearling vesicles7.