Mechanisms of COPII-Dependent Transport

目前的估计表明，在真核细胞中，25%-30%的翻译蛋白进入分泌途径。 在内质网(ER)中折叠和组装新生分泌蛋白后，外壳蛋白 复合体II(COPII)将折叠的货物分拣成运输囊泡，这些囊泡从内质网萌发，并靶向顺式- 高尔基车厢。遗传学和蛋白质组学方法已经确定了许多必需的成分 用于急诊室和高尔基建筑群之间的高效运输。然而，多样化的机制 分泌物被分类成COPII囊泡，然后萌发的囊泡如何靶向高尔基受体 细胞膜仍然很模糊。我们的研究项目应用多学科方法来定义分子 COPII囊泡和囊泡催化分泌货物依赖受体分选的机制 利用酵母菌作为模式生物，将其输送到高尔基复合体。我们开发了一种无细胞转运试验， 经过依赖COPIi的货物选择和囊泡的生物化学可分解阶段 发芽、Uso1p依赖的小泡拴系和SNARE蛋白依赖的膜融合。我们有 用分离的膜和纯化的可溶性分子重现了这些阶段。我的长期目标是 研究计划是通过对阶段和阶段的分析来阐明这些事件背后的催化机制。 用确定的蛋白质和脂肪组分进行特定的分析和重建实验。我们最近的进展 已经确定了跨膜货物受体(例如Erv26p和Erv29p)，它可以选择分泌蛋白进入 COPII囊泡的功能与内质网质量控制途径有关。此外，我们在以下方面取得了进展 在内质网向高尔基体运输的不同阶段，表征特定的脂质需求，包括PI(4)P 在下一个资金期，我们计划测试分子模型，以确定货物与出口受体的结合情况 调节；货物受体如何在内质网质量控制中发挥作用；特定蛋白质和脂类在内质网质量控制中的作用 组织ER出口站点和COPII萌芽；以及系留因素和PL(4)在向 高尔基情结。 相关性(见实例)； 这些实验目的是为了解决有关分泌蛋白质是如何 有选择地从ER输出，并被引导到高尔基建筑群。这一细胞内的运输步骤是 对细胞生长和调节至关重要。因此，这些研究是理解众多 与健康相关的问题，包括胆固醇调节、囊性纤维化和糖尿病。

Current estimates indicate that 25-30% of translated proteins enter the secretory pathway In eukaryotic cells. After folding and assembly of nascent secretory proteins in the endoplasmic reticulum (ER), the coat protein complex II (COPII) sorts folded cargo into transport vesicles that bud from the ER and are targeted to cis- Golgi compartments. Genetic and proteomic approaches have identified many of the components required for efficient transport between the ER and Golgi complex. However, the mechanisms by which diverse secretory cargo are sorted into COPII vesicles and how budded vesicles are then targeted to Golgi acceptor membranes remain obscure. Our research program applies a multidisciplinary approach to define molecular mechanisms that catalyze receptor-dependent sorting of secretory cargo into COPII vesicles and vesicular transport to the Golgi complex using yeast as a model organism. We exploit a cell-free transport assay that proceeds through the biochemically resolvable stages of COPIi-dependent cargo selection and vesicle budding, Uso1p-dependent vesicle tethering, and SNARE protein-dependent membrane fusion. We have reproduced these stages with isolated membranes and purified soluble molecules. The long-term goal of my research program is to elucidate the catalytic mechanisms undertying these events though analysis of stage- specific assays and reconstitution experiments with defined protein and lipid fractions. Our recent progress has identified transmembrane cargo receptors (e.g. Erv26p and Erv29p) that select secretory proteins into COPII vesicles and function with the ER quality control pathway. In addition, we have made progress in characterizing specific lipid requirements, including PI(4)P, in distinct stages of ER to Golgi transport, in the next funding period we plan test molecular models to determine how cargo binding to export receptors is regulated; how cargo receptors function in ER quality control; the role of specific proteins and lipid species in organizing ER export sites and COPII budding; and the role of tethering factors and Pl(4) in transport to the Golgi complex. RELEVANCE (See instnjctions); These experimental aims are designed to address fundamental questions on how secretory proteins are selectively exported from the ER and directed to the Golgi complex. This intracellular transport step is essential for cell growth and regulation. Therefore these studies are basic for understanding numerous health related issues including cholesterol regulation, cystic fibrosis and diabetes.