Molecular Mechanism of Sec1p-Regulated Membrane Fusion

Sec1p调控的膜融合的分子机制

• 批准号: 7002257
• 负责人: CHAVELA M CARR
• 金额: $ 21.23万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-05 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7002257
• 关键词: Molecular; Mechanism; Sec1p; Regulated; Membrane

DESCRIPTION (provided by applicant): Membrane fusion is a central theme in cell biology. For example, in order to enter the host, enveloped viruses use a specialized envelope protein to catalyze fusion of the viral and host cell membranes. The recognition that a similar mechanism is used for intracellular membrane fusion emerged from the observation that cellular proteins, called SNAREs, share structural similarity with the fusion-active conformation of the viral envelope proteins. Despite the inherent ability of SNARE proteins to fuse membranes, eukaryotic cells require at least a dozen other proteins for vesicle fusion at the plasma membrane. Our goal is to determine how these proteins work together to ensure accurate vesicle targeting and fusion. One of these proteins, Sec1p, is proposed to be essential for the assembly of SNAREs into a fusion-active complex that pins membranes together, for fusion. This model gains support from the recent structure of neuronal n-Sec1 in a complex with the SNARE Syntaxin-1A. Unexpectedly, predictions of this hypothesis do not hold when tested in S. cerevisiae, a model system for secretion studies. Based on the assembly model, an absence of Sec1p function is expected to block SNARE-complex assembly. Instead, levels of SNARE complexes in the loss-of-function secretory mutant, sec1-1 are unaltered. Furthermore, unlike the neuronal counterpart, yeast Sec1p has no observable affinity for the syntaxin protein, but instead binds to assembled SNARE complexes at sites of secretion. These conflicting observations indicate that our understanding of Sec1p-dependent vesicle fusion is currently limited and, therefore, requires further study. To address this problem, we propose (I) to map on a structure of yeast Sec1p the sites required for exocytosis, using a combination of genetics and X-ray crystallography, (ii) to test hypotheses for the mechanism of fusion regulation by reconstitution of Sec1p-dependent membrane fusion and (iii) to establish whether Sec1 p homologs share a common function in vesicle fusion, by defining the SNARE-binding and membrane-fusion properties of other yeast Sec1 p homologs including Sly1 p, a yeast Sec1 p homolog with syntaxin-binding properties similar to n-Sec1.

描述(申请人提供)：膜融合是细胞生物学的中心主题。例如，为了进入宿主，被包膜的病毒使用一种特殊的包膜蛋白来催化病毒和宿主细胞膜的融合。人们认识到，细胞内的膜融合也使用了类似的机制，这是因为观察到细胞蛋白质，称为SNARE，与病毒包膜蛋白的融合活性构象有相似的结构相似之处。尽管SNARE蛋白具有固有的融合膜的能力，但真核细胞至少需要十几种其他蛋白才能在质膜上进行囊泡融合。我们的目标是确定这些蛋白质如何协同工作，以确保准确的囊泡靶向和融合。这些蛋白质中的一种，Sec1p，被认为是将圈套组装成融合活性复合体的关键，该复合体将膜固定在一起进行融合。这一模型得到了最近神经元n-Sec1结构的支持，该结构与SNARE Synaxin-1A形成了一个复合体。出乎意料的是，这一假设的预测在酿酒酵母中进行测试时并不成立，酿酒酵母是一种用于分泌物研究的模型系统。基于装配模型，Sec1p功能的缺失有望阻碍复杂的装配。相反，功能丧失的分泌突变体sec1-1中的SNARE复合体的水平没有改变。此外，与神经元不同的是，酵母Sec1p对合成素蛋白没有明显的亲和力，而是在分泌部位与组装的SNARE复合体结合。这些相互矛盾的观察表明，我们对Sec1p依赖的囊泡融合的理解目前是有限的，因此需要进一步研究。为了解决这个问题，我们建议(I)结合遗传学和X射线结晶学，在酵母Sec1p的结构上定位胞吐所需的位点，(Ii)通过重建依赖于Sec1p的膜融合来测试融合调节机制的假说，以及(Iii)通过定义其他酵母Sec1p同源物的陷阱结合和膜融合特性，确定Sec1p同源物是否在囊泡融合中共享共同功能，包括Sly1p，一种具有与n-Sec1类似的Synaxin结合特性的酵母Sec1p同源物。