A NOVEL PROTEIN COMPLEX REQUIRED FOR ENDOSOMAL SORTING OF MEMBRANE TRANSPORTERS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 内吞作用后分选到溶酶体是下调细胞表面受体和转运蛋白丰度的关键机制。在全基因组化学遗传筛查中，我们鉴定了一种特性不佳的酿酒酵母蛋白，这是许多细胞表面蛋白内体分选所必需的。在高等物种中也发现了潜在的同源物。缺乏该基因的酵母菌株的表型表明内体小泡的融合/对接存在缺陷。该蛋白定位于细胞质和内体。在凝胶过滤实验中，细胞质池呈现为均一的650 kDa的复合体。我们假设胞液复合体可能是内体结合形式的亚复合体。因此，了解650-kD可溶复合体的组成可能有助于更好地理解内体融合和对接的机制。我们建议用质谱学方法研究TAP-纯化络合物的组成。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Endocytosis followed by sorting to the lysosome is a key mechanism for downregulating the abundance of cell surface receptors and transporters. In a genome-wide chemical genetic screen we have identified a poorly characterized S. cerevisiae protein that is required for endosomal sorting of many cell surface proteins. Potential homologs in higher species have also been identified. Phenotype of the yeast strain lacking this gene suggests a defect in fusion/docking of endosomal vesicles. The protein localizes to the cytoplasm as well as endosomes. The cytoplasmic pool appears as a homogenous, 650-kDa complex in gel filtration experiments. We hypothesize that the cytosolic complex may be a subcomplex of the endosome-bound form. Thus, understanding the composition of the 650-kD soluble complex may lead to a better understanding of the mechanism of endosome fusion and docking. We propose to study the composition of the TAP-purified complex by mass spectrometry.