INTRACELLULAR TRANSPORT--THE MANNOSE/PHOSPHATE RECEPTOR

细胞内转运——甘露糖/磷酸盐受体

• 批准号: 2443991
• 负责人: Suzanne R Pfeffer
• 金额: $ 25.67万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2443991
• 关键词: Golgi apparatus; carbohydrate receptor; fatty acylation; guanosinetriphosphatases; intracellular transport; laboratory mouse; laboratory rabbit; lysosomes; mannose; mannose 6 phosphate; membrane reconstitution /synthesis; mutant; myristates; phosphates; protein transport; vesicle /vacuole

During intracellular transport, proteins destined for the plasma membrane, secretory vesicles and lysosomes must be sorted from one another within the Golgi complex, and sent to their appropriate addresses. The long term goal of this research is to elucidate the molecular mechanisms by which proteins are targeted to specific and distinct intracellular destinations. We study the targeting of proteins to lysosomes. Soluble lysosomal enzymes acquire a mannose 6-phosphate tag within the Golgi complex; intracellular receptors (MPRs) recognize these proteins, and facilitate their transport to lysosomes. We have reconstituted the recycling of MPRs from late endosomes to the trans Golgi network (TGN) in crude extracts. Our most significant accomplishment of the previous funding period was the discovery that rab9, a small ras-like GTPase, facilitates this transport reaction, both in vitro and in vivo. The present application describes experiments designed to continue our molecular dissection of MPR trafficking. The specific aims of this proposal are: 1. Elucidate the mechanism by which rab9 protein is localized specifically to late endosomes using an in vitro system which reconstitutes organelle-specific localization of rab 9 protein in vitro. 2. Dissect our in vitro assay into vesicle budding and targeting subreactions and establish a functional connection between rab recruitment and subsequent MPR transport from late endosomes to the TGN. 3. Test the validity of the SNARE Hypothesis for endosome-TGN transport by establishing whether known SNARE constituents participate in MPR recycling from late endosomes to the TGN in vitro. Use a functional assay for rab9 protein to attempt to link the SNARE machinery with rab protein function. 4. Investigate the role of ARF in endosome-to-TGN transport to aid in the identification of the putative coat proteins and transport vesicles (or tubules) responsible for this transport step. It is hoped that these experiments will provide new molecular detail as to the mechanism by which proteins are delivered from one membrane-bound compartment to another.

在细胞内转运过程中，蛋白质被送往血浆 膜，分泌囊泡和溶酶体必须从一个分类 另一个在高尔基复合体内，并发送到他们适当的 地址. 这项研究的长期目标是阐明 蛋白质靶向特定的和 不同的细胞内目的地。 我们研究蛋白质的靶向 到溶酶体。 可溶性溶酶体酶获得甘露糖6-磷酸 高尔基复合体内的标签;细胞内受体（MPR）识别 这些蛋白质，并促进其运输到溶酶体。 我们有 重建了MPR从晚期内体到反式内体的再循环， 粗提物中的高尔基体网络（TGN）。 我们最重要 上一个融资期的成就是发现， rab 9是一个小的ras样GTd 3，促进了这种转运反应， 在体外和体内。 本申请描述了实验 旨在继续我们对MPR贩运的分子解剖 的 这项建议的具体目标是： 1.阐明rab 9蛋白定位的机制 特别是晚期核内体， 在体外重建rab 9蛋白的细胞器特异性定位。 2.将我们的体外试验分解为囊泡出芽和靶向 并在RAB之间建立功能连接 募集和随后MPR从晚期内体转运至TGN。 3.检验内体-TGN转运的陷阱假说的有效性 通过确定已知SNARE成分是否参与MPR 在体外从晚期内体再循环到TGN。 使用功能性 尝试将SNARE机制与rab连接的rab 9蛋白的测定 蛋白质功能 4.研究ARF在内体至TGN转运中的作用，以帮助 鉴定推定的外壳蛋白和运输囊泡（或 管）负责该运输步骤。 希望这些实验将提供新的分子细节， 蛋白质从一个膜结合的 车厢到另一个。