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{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3216505","sourcedb":"PMC","sourceid":"3216505","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3216505","text":"Discussion\nIn vitro reconstitution experiments with artificial lipid bilayers, COPII proteins and non-hydrolysable GTP analog can be performed in different ways, involving different degrees of mechanical processing and separation: (i) Reactions of GUVs with COPII and GMP-PNP without any centrifugation step yielded very long vesicle strings, shorter strings and some single vesicles (this work, Fig. 3a–h). (ii) Reactions of LUVs with COPII and GMP-PNP visualized by EM after chemical fixation and sedimentation showed buds, single vesicles and short strings of vesicles2. (iii) Reactions of LUVs with COPII and GMP-PNP that were ultracentrifuged on a gradient contained a homogeneous fraction of small coated vesicles8. Together, these observations suggest that the vesicle strings formed with non-hydrolysable GTP analog may be precursors to single vesicles. Moreover, in vitro scission appears to be promoted by mechanical force.\nIt was previously noted that COPII vesicles produced in vitro have a similar appearance and diameter as native ones8. By keeping sample manipulation minimal, we have now been able to observe extended constricted tubules in vitro (Fig. 3a–h) and found that they exhibit strikingly similar shapes and diameters to those seen in cells (Fig. 3i, ref. 141516). In cells, single vesicles, “dumb-bell shapes”, “tubular membranes with vesicular-shaped endings”16 and short “beaded necklaces”15 have been observed. Networks of longer vesicle strings with occasional branches were reported when GTP hydrolysis was blocked by the Sar1(H79G) mutation. This led to the suggestion that “budding continues in the absence of GTP hydrolysis, but […] vesicles fail to complete separation from one another.”15\nOur in vitro reconstitution results strongly parallel these in vivo observations. With nonhydrolysable GMP-PNP, extended necklace-like vesicle strings with various degrees of constrictions and occasional branches were observed (Fig. 3a–h). Vesicle pairs resembled dumb-bells. Nearly tubular sections were connected to vesicular-shaped endings. This diverse set of lipid structures that carry some level of COPII protein coat is consistent with the possibility that extended and large cargo is transported in tubular sections (as depicted schematically in Fig. 3g) or in ‘super-sized' COPII bulges.\nIn vitro, vesicle strings developed to remarkable dimensions of dozens of connected vesicles, several micrometers in total length (Fig. 2b–d), which supports the notion of Bannykh et al.15 that vesicle separation fails in the absence of GTP hydrolysis. To exclude extraneous effects of non-hydrolysable GTP analogs, we used hydrolysable GTP and replaced wtSar1p by Sar1p(H77L) (Fig. 2j). The intrinsic GTPase activity of this mutant is similarly low as that of wtSar1p, but stimulation of the GTPase activity by the Sec23/24p complex is reduced from about 9-fold to 2-fold3. The lowered GTPase activity of the mutant in the presence of the full COPII complex proved sufficient for the formation of rigid, multibudded extensions (Fig. 2j, Movie S3, Fig. 3k), as seen with the non-hydrolysable GTP analog.\nOur in vitro data illustrates that a block of GTP hydrolysis generates unfissioned precursors of COPII vesicles. This result leaves the question open how GTP hydrolysis and fission are controlled. Incubations containing GTP instead of GMP-PNP on artificial giant liposomes did not generate separate vesicles on an appreciable scale. The less dramatic GUV morphology change observed with COPII/GTP (Fig. 2h,i) is consistent with the finding that, with GTP, lipid bilayers fail to retain the membrane-bound COPII coat as measured in light scattering experiments17 and during density gradient sedimentation1. Coat stability was enhanced by the nucleotide exchange factor Sec12ΔCp compared to the less active nucleotide exchange facilitated by lowering Mg2+ with EDTA1. Presumably, Sec12ΔCp allows multiple rounds of nucleotide hydrolysis and exchange. In agreement, COPII/GTP reactions had a stronger morphological effect on GUVs in the presence of Sec12ΔCp than with EDTA (Fig. 2h vs. 2i), whereas no difference between EDTA and Sec12ΔCp was observed in incubations containing GMP-PNP (Fig. 2c vs. 2b).\nObviously, uninhibited large-scale disintegration of a lipid bilayer into vesicles is not desirable in cells. A complex interplay of factors may be necessary to regulate vesicle fission processes, as suggested in a study of COPI assembly on GUVs, where membranes also failed to undergo fission18. Regulation of COPII vesicle fission in vivo may be provided through the localization of specific factors (Sec16p and membrane-anchored Sec12p, for instance) and by triggering events, such as the sorting of cargo proteins into buds, as suggested by Tabata et al.19. The regulation of Sar1p-mediated GTP hydrolysis in relation to membrane fission requires further investigation.\nOne hypothesis for a fission pathway9 postulates that GTP hydrolysis leads to unbinding of the Sar1 amphipathic helix from the membrane, resulting in packing defects. Such defects should lead to rapid changes in membrane morphology to fill the gaps and avert the energetically unfavorable exposure of the hydrophobic lipid chains to water. To preclude simple contraction (i.e., shortening of the lipid tubule) as a path of relaxation, a separation force may be needed. Separation forces can be static, i.e. maintaining the length of the protrusion, or dynamic, i.e. actively extending the protrusion. Static separation forces, applied artificially by attachments to an EM grid, have been reported to support fission of dynamin-mediated tubular constrictions2021. In vivo, dynamic separation forces may be exerted by motor proteins and cytoskeleton, for example, by dynactin/dynein coupling to Sec23p22. Here, we observed that tubules formed by COPII were, despite their constrictions, globally rigid over several micrometer (Fig. 3b, Movie S2), demonstrating that the COPII scaffold conveys rigidity and directionality including at the necks between the bulges. The coat may thus itself act as an ‘exoskeleton', providing a static separation force between the bulges and obviating the need for cytoskeleton involvement in simple COPII budding.\nFurthermore, coat completion may actively contribute to fission. In the absence of Sar1p and membranes, the coat has a propensity to assemble into spherical structures, which have positive curvature in both principal directions1223. This propensity may help to promote the transition from tubular membranes, which have positive curvature in only one direction, to vesicles, which have positive curvature in both directions (Fig. 3c).","divisions":[{"label":"title","span":{"begin":0,"end":10}},{"label":"p","span":{"begin":11,"end":933}},{"label":"p","span":{"begin":934,"end":1724}},{"label":"p","span":{"begin":1725,"end":2322}},{"label":"p","span":{"begin":2323,"end":3126}},{"label":"p","span":{"begin":3127,"end":4227}},{"label":"p","span":{"begin":4228,"end":4901}},{"label":"p","span":{"begin":4902,"end":6245}}],"tracks":[{"project":"2_test","denotations":[{"id":"22355536-16122427-137225138","span":{"begin":571,"end":572},"obj":"16122427"},{"id":"22355536-9568718-137225139","span":{"begin":719,"end":720},"obj":"9568718"},{"id":"22355536-9568718-137225140","span":{"begin":1049,"end":1050},"obj":"9568718"},{"id":"22355536-17981132-137225141","span":{"begin":1281,"end":1283},"obj":"17981132"},{"id":"22355536-8858160-137225142","span":{"begin":1281,"end":1285},"obj":"8858160"},{"id":"22355536-16531996-137225143","span":{"begin":1281,"end":1287},"obj":"16531996"},{"id":"22355536-16531996-137225144","span":{"begin":1386,"end":1388},"obj":"16531996"},{"id":"22355536-8858160-137225145","span":{"begin":1417,"end":1419},"obj":"8858160"},{"id":"22355536-8858160-137225146","span":{"begin":1722,"end":1724},"obj":"8858160"},{"id":"22355536-8858160-137225147","span":{"begin":2509,"end":2511},"obj":"8858160"},{"id":"22355536-9756629-137225148","span":{"begin":2895,"end":2896},"obj":"9756629"},{"id":"22355536-11389436-137225149","span":{"begin":3686,"end":3688},"obj":"11389436"},{"id":"22355536-15457212-137225150","span":{"begin":3730,"end":3731},"obj":"15457212"},{"id":"22355536-15457212-137225151","span":{"begin":3890,"end":3891},"obj":"15457212"},{"id":"22355536-18974217-137225152","span":{"begin":4521,"end":4523},"obj":"18974217"},{"id":"22355536-19763084-137225153","span":{"begin":4786,"end":4788},"obj":"19763084"},{"id":"22355536-16782321-137225154","span":{"begin":4938,"end":4939},"obj":"16782321"},{"id":"22355536-15450295-137225155","span":{"begin":5659,"end":5661},"obj":"15450295"},{"id":"22355536-19903475-137225156","span":{"begin":5659,"end":5663},"obj":"19903475"},{"id":"22355536-15580264-137225157","span":{"begin":5801,"end":5803},"obj":"15580264"},{"id":"22355536-18692470-137225158","span":{"begin":6473,"end":6475},"obj":"18692470"},{"id":"22355536-17604721-137225159","span":{"begin":6473,"end":6477},"obj":"17604721"}],"attributes":[{"subj":"22355536-16122427-137225138","pred":"source","obj":"2_test"},{"subj":"22355536-9568718-137225139","pred":"source","obj":"2_test"},{"subj":"22355536-9568718-137225140","pred":"source","obj":"2_test"},{"subj":"22355536-17981132-137225141","pred":"source","obj":"2_test"},{"subj":"22355536-8858160-137225142","pred":"source","obj":"2_test"},{"subj":"22355536-16531996-137225143","pred":"source","obj":"2_test"},{"subj":"22355536-16531996-137225144","pred":"source","obj":"2_test"},{"subj":"22355536-8858160-137225145","pred":"source","obj":"2_test"},{"subj":"22355536-8858160-137225146","pred":"source","obj":"2_test"},{"subj":"22355536-8858160-137225147","pred":"source","obj":"2_test"},{"subj":"22355536-9756629-137225148","pred":"source","obj":"2_test"},{"subj":"22355536-11389436-137225149","pred":"source","obj":"2_test"},{"subj":"22355536-15457212-137225150","pred":"source","obj":"2_test"},{"subj":"22355536-15457212-137225151","pred":"source","obj":"2_test"},{"subj":"22355536-18974217-137225152","pred":"source","obj":"2_test"},{"subj":"22355536-19763084-137225153","pred":"source","obj":"2_test"},{"subj":"22355536-16782321-137225154","pred":"source","obj":"2_test"},{"subj":"22355536-15450295-137225155","pred":"source","obj":"2_test"},{"subj":"22355536-19903475-137225156","pred":"source","obj":"2_test"},{"subj":"22355536-15580264-137225157","pred":"source","obj":"2_test"},{"subj":"22355536-18692470-137225158","pred":"source","obj":"2_test"},{"subj":"22355536-17604721-137225159","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93c1","default":true}]}]}}