RESUMO
Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/αSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 can drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE that face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.
Assuntos
Adenosina Trifosfatases/metabolismo , Fusão de Membrana/genética , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatases/genética , Comunicação Celular , Fusão de Membrana/fisiologia , Domínios Proteicos , Proteínas SNARE/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
To better understand animal cell plasma membranes, we studied simplified models, namely four-component lipid bilayer mixtures. Here we describe the domain size transition in the region of coexisting liquid-disordered (Ld) + liquid-ordered (Lo) phases. This transition occurs abruptly in composition space with domains increasing in size by two orders of magnitude, from tens of nanometers to microns. We measured the line tension between coexisting Ld and Lo domains close to the domain size transition for a variety of lipid mixtures, finding that in every case the transition occurs at a line tension of â¼0.3 pN. A computational model incorporating line tension and dipole repulsion indicated that even small changes in line tension can result in domains growing in size by several orders of magnitude, consistent with experimental observations. We find that other properties of the coexisting Ld and Lo phases do not change significantly in the vicinity of the abrupt domain size transition.