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来理解溶酶体是如何形成的以及它是如何介导膜蛋白降解的。一个促进溶酶体和溶酶体相关细胞器生物发生的运输途径是由接头蛋白-3 （AP-3）囊泡外壳蛋白复合物控制的。在人类中，失去功能性AP-3会导致黑色素生成和血小板功能的缺陷，而没有AP-3的小鼠也会出现神经缺陷。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），并允许它们绕过泡前/内体隔室。我们已经进行了基因筛选，以确定AP-3运输途径的其他蛋白质，现在将分析这些蛋白质的功能作用。通过泡前/内体隔室的蛋白质会经历不同的分选过程。在这里，它们可以被纳入核内体的管腔膜，然后在液泡内部传递和降解。这种机制确保了膜蛋白的细胞质和管腔结构域的破坏。并不是所有通过核内体的蛋白质都会被腔囊泡降解。我们发现，当泛素附着在膜蛋白上时，它作为一个特定的分选信号，进入内体管腔囊泡。我们还确定了一个假定的泛素分类受体，它将识别注定要降解的蛋白质。这种受体复合体也在将从核内体中回收的蛋白质分选回高尔基体中起作用。我们现在将在分子水平上表征这种候选受体的功能，以确定它是如何实现其独特和多种功能的。这一分析将通过体外重建管腔囊泡形成过程和蛋白质分选得到支持。

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.