Molecular Mechanism of Sec1p-Regulated Membrane Fusion

Sec1p调控的膜融合的分子机制

• 批准号: 6533547
• 负责人: CHAVELA M CARR
• 金额: $ 26.92万
• 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/6533547
• 关键词: Saccharomyces cerevisiae; X ray crystallography; binding sites; cell membrane; colorimetry; exocytosis; fluorescence resonance energy transfer; genetic screening; immunoprecipitation; intracellular transport; membrane fusion; membrane proteins; protein protein interaction; protein purification; protein reconstitution; protein structure function; syntaxin

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（由申请人提供）：膜融合是细胞生物学的中心主题。例如，为了进入宿主，包膜病毒使用专门的包膜蛋白来催化病毒和宿主细胞膜的融合。类似机制用于细胞内膜融合的认识来自于观察到称为SNARE的细胞蛋白与病毒包膜蛋白的融合活性构象具有结构相似性。尽管SNARE蛋白具有融合膜的固有能力，但真核细胞需要至少十几种其他蛋白质才能在质膜上融合囊泡。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。其中一种蛋白质Sec 1 p被认为是将SNARE组装成融合活性复合物所必需的，该复合物将膜固定在一起以进行融合。该模型得到了最近神经元n-Sec 1与SNARE Syntaxin-1A复合物结构的支持。出乎意料的是，当在S中检验时，这一假设的预测不成立。cerevisiae，用于分泌研究的模型系统。基于组装模型，预期Sec 1 p功能的缺失将阻止SNARE复合物组装。相反，SNARE复合物在功能丧失的分泌突变体sec 1 -1中的水平没有改变。此外，与神经元对应物不同，酵母Sec 1 p对突触融合蛋白没有可观察到的亲和力，而是在分泌位点与组装的SNARE复合物结合。这些相互矛盾的观察结果表明，我们的理解Sec 1 p依赖囊泡融合目前是有限的，因此，需要进一步的研究。为了解决这个问题，我们建议（I）映射酵母Sec 1 p的结构上的胞吐作用所需的网站，使用遗传学和X射线晶体学的组合，（ii）测试通过重建Sec 1 p依赖的膜融合融合的融合调节机制的假设，（iii）建立Sec 1 p同源物是否在囊泡融合中共享共同的功能，通过定义其他酵母Sec 1 p同系物的SNARE结合和膜融合特性，包括Sly 1 p，一种具有与n-Sec 1相似的突触融合蛋白结合特性的酵母Sec 1 p同系物。