Alternative Protein Sorting in the Trans-Golgi Network

描述(申请人提供)：我们正在使用酿酒酵母作为一个模型系统，以了解溶酶体是如何形成的，以及它是如何介导膜蛋白的降解的。促进溶酶体和溶酶体相关细胞器生物发生的一条运输途径是由接头蛋白-3(AP-3)囊泡外壳蛋白复合体控制的。在人类中，功能AP-3的缺失会导致黑素合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运输到液泡(溶酶体)，并允许它们绕过泡前/内体隔室的运输。我们已经进行了一次基因筛查，以确定参与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。确实通过泡前/内膜间隔的蛋白质受到不同的分选事件的影响。在这里，它们可以被结合到内体的管腔膜中，这些膜又被输送到液泡内部并在液泡内部降解。这一机制确保了膜蛋白的胞浆结构域和腔结构域的破坏。并不是所有通过内体的蛋白质都以腔小泡为目标进行降解。我们发现，当泛素附着在膜蛋白上时，它作为一种特异性的分选信号被整合到内体的腔小泡中。我们还确定了泛素的一个假定的分类受体，它可以识别注定要降解的蛋白质。这种受体复合体还起到分选蛋白质的作用，这些蛋白质从内体循环回到高尔基体。我们现在将在分子水平上表征这个候选受体的功能，以确定它如何执行其独特的和多重的功能。这一分析将通过在体外重建腔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.