Results

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.

COPII forms rigid tubules
When giant liposomes were incubated with the full COPII coat (Sar1p, Sec23/24p and Sec13/31p) in the presence of GTP, confocal microscopy showed a heterogeneous picture, consisting of intact GUVs, ‘granulated' GUVs, tubules, small and larger vesicles and aggregated membranes (Fig. 2h,i). A biochemical budding assay that involved sucrose gradient centrifugation did not show a distinguishable budded vesicle fraction in incubations containing GTP18. A clear fraction of budded vesicle was only observed with non-hydrolysable GMP-PNP, suggesting that the minimal COPII system used in vitro lacks factors that govern the spatial and temporal coordination of fission. We therefore adopted the GMP-PNP condition that produces robust vesicle formation in the biochemical assay for use in our GUV assay.
The generally accepted picture of budding depicts vesicles as forming from single-vesicle-buds910. Taken together with the positive budding results by Matsuoka et al.8, we expected to obtain separated COPII vesicles in incubations of GUVs with COPII proteins and GMP-PNP. Because COPII vesicles are below optical resolution, we expected a hazy fluorescence background in the space outside the mother liposomes. This was not observed. Instead, prominent straight and rigid extensions were formed (Fig. 2b, Movie S2). Some GUVs appeared fully tubulated. Others were densely covered with COPII-coated extensions that remained connected to the GUV, yielding a ‘star-fish' like appearance. Some extensions were more than 10 µm long. Notably, the persistence length was much greater than the length of the tubules. Fluorescence intensity was fairly uniform, indicating that all tubular extensions had a uniform, defined thickness.

COPII shows a preference for high curvature structures
The COPII protein coat was clearly localized on the rigid extensions, as seen from a comparison of the fluorescence signals of the lipid and the Sec13/31 coat (Fig. 2c). In fact, more COPII was bound to the highly curved extensions than to the low curvature GUV surface. This implies that the COPII complex plays a role in remodeling the lipid bilayers into highly curved structures or that it preferentially binds highly curved surfaces, presumably stabilizing them.

The COPII proteins act jointly in membrane deformation
No differences in rigid tubulation were observed if nucleotide exchange on Sar1p was achieved by the addition of EDTA to lower Mg2+ (e.g., Fig. 2b) or by the addition of Sec12ΔCp (e.g., Fig. 2c). The presence of the N-terminal amphipathic helix of Sar1p was required for COPII recruitment (Fig. 2g). At a reduced concentration (one-tenth) of Sar1p, tubules nonetheless formed, albeit with a lower yield (Fig. 2f). We also noted that the outer Sec13/31p coat complex was required to observe separated tubules. When Sec13/31p was omitted as in reconstitution experiments of pre-budding complexes11, the GUVs aggregated, suggesting exposed hydrophobic contacts on the incomplete COPII coat (Fig. 2e vs. 2d, Fig. S2). These observations confirm the importance of Sar1p and the coordinated action of the COPII proteins.

Electron microscopy reveals multibudded tubules
Because the diameter of COPII-coated tubules is below the optical resolution of confocal microscopy, we used EM for a more detailed examination. Cryo-EM revealed that the COPII-coated tubules carry symmetric constrictions at regular intervals (Fig. 3). In other words, they consist of unfissioned vesicles, like beads on a string. The size of the connected vesicles in cryo-EM was rather homogeneous (Fig. 3a–h), although variations occurred, for instance at the end of a string (Fig. 3e). Apart from the prominent connected protein-coated vesicles, single vesicles as well as larger liposomes were observed (Fig. 3a,d–h).
Most beads-on-a-string vesicles were linearly connected, with occasional branches. Clover shapes, which appeared as branches, actually comprised two crossed-over tubules (Fig. 3a,b,f). Some of the constricted tubules deposited on the carbon support of the EM grid were up to several micrometers long and completely straight (Fig. 3f), whereas others apparently became entangled during the preparation (Fig. 3a). Except for the constrictions, tubules were mostly uniform in thickness. The appearance of the tubules in cryo-EM hence agreed well with their appearance by confocal microscopy. Furthermore, the cryo-EM images clearly showed protein coat on the lipid bilayer (Fig. 3b,d,e, arrows), even at the ‘necks' between bulges, explaining the great persistence length. Whereas the typical strings of vesicles were all coated with protein, some of the larger liposomes appeared coated (Fig. 3d,e arrows) and others not (Fig. 3e, arrowhead). This observation seems to suggest that - despite the preference of the COPII coat for high-curvature structures seen by fluorescence – COPII proteins have some flexibility for binding to lower curvature membranes. The ability of the COPII coat to bind to membranes of different curvatures merits further investigation to determine how COPII cages form on ‘oversized' cargo such as procollagen and chylomicrons1213.