INTRACELLULAR TRANSPORT--THE MANNOSE/PHOSPHATE RECEPTOR

在细胞内运输过程中，流向血浆的蛋白质 膜、分泌小泡和溶酶体必须从一个中分离出来 另一个在高尔基建筑群内，并送到他们相应的 地址。这项研究的长期目标是阐明 蛋白质靶向特定基因和蛋白质的分子机制 不同的细胞内目的地。我们研究了蛋白质的靶向性。 到溶酶体。可溶性溶酶体酶可获得甘露糖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，并促进它们的运输到溶酶体。我们有 重组了MPR从晚内体到反式内体的循环 粗提物中的高尔基网络(TGN)。我们最重要的 上一个资金期的完成是发现 Rab9，一种类似ras的小GTP酶，促进了这种运输反应，两者 在体外和体内。本申请描述了实验 旨在继续我们对MPR走私的分子解剖。这个 这项建议的具体目标是： 1.阐明Rab9蛋白的定位机制 特别是使用体外系统对晚期内小体进行识别 重组Rab 9蛋白在体外的细胞器特异性定位。 2.分析我们的体外实验中的囊泡萌发和靶向 亚反应和建立Rab之间的功能联系 募集和随后的MPR运输从晚期内体到TGN。 3.检验内体-TGN转运的陷阱假说的有效性 通过确定已知的陷阱成分是否参与MPR 在体外，从较晚的内体循环到TGN。使用功能组件 检测Rab9蛋白以尝试将诱捕机制与RAb联系起来 蛋白质的功能。 4.研究ARF在内体到TGN转运中的作用，以帮助 推测的外壳蛋白和运输小泡(或 小管)负责这一运输步骤。 希望这些实验将提供新的分子细节，如 到蛋白质从一个膜结合传递的机制 换到另一个车厢。

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.