Molecular mechanisms of cargo capture into ER-derived transport vesicles

内质网衍生运输囊泡中货物捕获的分子机制

• 批准号: 8598890
• 负责人: Elizabeth A. Miller
• 金额: $ 30.58万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598890
• 关键词: Adaptor Signaling Protein; Anemia; Animal Model; Biochemical; Biogenesis; Bypass; Carrier Proteins; Cell physiology; Cells; Charge; Cytoplasmic Protein; Defect; Development; Disease; Endoplasmic Reticulum; Ensure; Environment; Eukaryotic Cell; Event; Genetic; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hemophilia A; Individual; Lipid Bilayers; Mediating; Membrane; Membrane Proteins; Modeling; Molecular; Molecular Probes; Monitor; Morphology; Mutation; Organelles; Pathway interactions; Play; Process; Property; Protein Secretion; Proteins; Proteome; Regulation; Research Proposals; Role; Saccharomyces cerevisiae; Site; Sorting - Cell Movement; Specificity; Structure; Transport Vesicles; Vesicle; Work; base; biophysical properties; developmental disease; environmental change; human disease; in vivo; in vivo imaging; mutant; novel; polypeptide; premature; protein folding; protein transport; public health relevance; secretory protein; segregation; tool; trafficking; yeast genetics

DESCRIPTION (provided by applicant): Efficient and accurate protein secretion is a fundamental process that plays a pivotal role in the ability of al eukaryotic cells to function, grw and communicate. Fully one-third of the eukaryotic proteome is targeted to the membrane compartments that comprise the secretory pathway. These proteins must be faithfully delivered to these organelles after synthesis and folding in the endoplasmic reticulum (ER). We study the biogenesis of ER-derived transport vesicles, aiming to uncover the rules that govern efficient capture of cargo proteins and the biophysical basis of membrane transformation events that give rise to spherical protein carriers. We use the model organism, Saccharomyces cerevisiae, to study this problem using a combination of genetic, biochemical and in vivo imaging approaches. Vesicle formation encompasses three fundamental processes: deformation of the donor membrane into a spherical transport carrier; selective recruitment of cargo proteins into the nascent bud; and scission of the membrane to release the vesicle. Traditionally, these processes were thought to be driven by discrete functions of the COPII coat, but our recent findings suggest a more intimate connection between the different events that conspire to yield a vesicle. For example, lumenally-oriented cargo proteins likely oppose the action of the COPII coat in deforming the lipid bilayer; the specific cargo composition at individual ER exit sites can thus directly impact the efficiency of vesicle formation by altering membrane properties. Furthermore, the GTP cycle of the coat, which controls coat assembly, vesicle scission and coat shedding, is regulated in part by a partnership between the cargo adaptor protein, Sec24, and the COPII accessory protein, Sec16, implicating cargo proteins in modulation of the GTP cycle. Taken together, these new findings place the cargo molecules as central players in the process of vesicle formation in vivo, which makes sense if we consider that vesicle formation must be an adaptable process that permits cells to respond to the changes in specific cargo burdens associated with changing environmental and developmental conditions. The current research proposal consists of two specific aims. (1) To unravel the complexity of the GTP cycle of the COPII coat, probing the impact of alterations to the GTPase activity of the coat and elucidating whether cargo plays a direct role in this process. (2) To characterize the influence of cargo proteins on membrane biophysical properties that impact the ability of the COPII coat to deform the ER membrane into transport vesicles. Ultimately, a more detailed understanding of this fundamental eukaryotic process will have important implications in the many aspects of human disease that are impacted by defects in protein traffic within the secretory pathway.

描述（由申请人提供）：有效而准确的蛋白质分泌是一个基本过程，在Al真核细胞发挥作用，GRW和交流的能力中起着关键作用。完全三分之一的真核蛋白质组针对构成分泌途径的膜室。这些蛋白质必须忠实地递送到内质网（ER）中的合成和折叠后。我们研究了ER衍生的运输囊泡的生物发生，旨在发现有效捕获货物蛋白的规则以及膜转化事件的生物物理基础，这些膜转化事件引起了球形蛋白载体。我们使用模型生物酿酒酵母使用遗传，生化和体内成像方法的组合来研究这个问题。囊泡的形成包括三个基本过程：供体膜变形为球形运输载体；选择性募集货物蛋白进入新生的芽；和膜的分裂以释放囊泡。传统上，这些过程被认为是由COPII外套的离散功能驱动的，但是我们最近的发现表明，在共同产生囊泡的不同事件之间建立了更亲密的联系。例如，面向液体的货物蛋白可能反对Copii涂层变形脂质双层的作用。单个ER出口站点的特定货物组成可以 因此，直接通过改变膜特性来直接影响囊泡形成的效率。此外，涂层的GTP循环控制着外套组件，囊泡分裂和涂层，部分受到货物适配器蛋白，SEC24和COPII辅助蛋白SEC16之间的合作伙伴关系，这意味着货物蛋白在GTP周期的调节调节中。综上所述，这些新发现将货物分子作为囊泡形成过程中的核心参与者，如果我们认为囊泡形成必须是一个适应性的过程，这是有道理的，该过程允许细胞应对与环境和发育条件的变化相关的特定货物负担的变化。当前的研究建议包括两个具体目标。 （1）揭示了Copii涂层的GTP循环的复杂性，探测了改变对涂层GTPase活性的影响，并阐明了货物是否在此过程中起着直接作用。 （2）表征货物蛋白对膜生物物理特性的影响，这些特性会影响Copii外套将ER膜变形为运输囊泡的能力。最终，对这一基本真核过程的更详细的了解将对人类疾病的许多方面具有重要意义，这些方面受到分泌途径中蛋白质流量缺陷的影响。