Alternative Protein Sorting in the Trans-Golgi Network

描述（由申请人提供）：我们使用酿酒酵母作为模型系统，以了解溶酶体是如何形成的，以及它是如何介导膜蛋白降解的。一种有助于溶酶体和溶酶体相关细胞器的生物发生的运输途径由衔接蛋白-3（AP-3）囊泡外壳蛋白复合物控制。在人类中，功能性AP-3的丧失导致黑素生成和血小板功能的缺陷，而没有AP-3的小鼠也显示出神经缺陷。在酵母中，AP-3途径将一部分蛋白质从高尔基体转运到液泡（溶酶体），并允许它们绕过前液泡/内体区室。我们已经进行了遗传筛选，以确定其他蛋白质，有助于AP-3运输途径，现在将分析这些蛋白质的功能作用。蛋白质，通过prevacuolar/内体隔室旅行是受到不同的排序事件。在这里，它们可以被整合到内体的腔膜中，然后在液泡内部输送和降解。这种机制确保了膜蛋白的胞质和内腔结构域的破坏。并非所有穿过内体的蛋白质都靶向内腔囊泡进行降解。我们已经发现，当泛素连接到膜蛋白，它作为一个特定的分选信号纳入内体腔囊泡。我们还确定了一个假定的泛素分选受体，它将识别注定要降解的蛋白质。这种受体复合物也在分选蛋白质中起作用，这些蛋白质从内体再循环回到高尔基体。我们现在将在分子水平上描述这种候选受体的功能，以确定它如何执行其独特的多种功能。该分析将通过重建体外腔囊泡形成和蛋白质分选的过程来支持。

DESCRIPTION (provided by applicant): We are using Saccharomyces cerevisiae as a model system for understanding how the lysosome is formed and how it mediates the degradation of membrane proteins. One trafficking pathway that contributes to the biogenesis of lysosomes and lysosome-related organelles is controlled by the Adaptor Protein-3 (AP-3) vesicle coat protein complex. In humans, loss of functional AP-3 causes defects in melanogenesis and platelet function and mice without AP-3 also show neurological defects. In yeast, the AP-3 pathway transports a subset of proteins from the Golgi apparatus to the vacuole (lysosome) and allows them to bypass transit through prevacuolar/endosomal compartments. We have conducted a genetic screen to identify other proteins that contribute to the AP-3 trafficking pathway and will now analyze the functional role of these proteins.Proteins that do travel through the prevacuolar/endosomal compartment are subject to a different sorting event. Here they can be incorporated into lumenal membranes of the endosome that are in turn delivered and degraded in the interior of the vacuole. This mechanism ensures the destruction of both cytosolic and lumenal domains of membrane proteins. Not all proteins that travel through the endosome are targeted to lumenal vesicles for degradation. We have found that when Ubiquitin is attached to membrane proteins, it acts as a specific sorting signal for incorporation into endosomal lumenal vesicles. We have also identified a putative sorting receptor for Ubiquitin, which would recognize proteins fated for degradation. This receptor complex also plays a role in sorting proteins that recycle out of endosomes back to the Golgi apparatus. We will now characterize the function of this candidate receptor at the molecular level to define how it carries out its distinct and multiple functions. This analysis will be buttressed by reconstituting the process of lumenal vesicle formation and protein sorting in vitro.