PtdIns 4-Kinase Regulation of Protein Sorting in the Golgi Apparatus

描述（由申请人提供）：高尔基体有两个主要功能：糖蛋白和糖脂的生物合成，以及分选。这些功能是真核细胞基本结构的基础，因此了解高尔基体的功能是至关重要的。此外，许多人类疾病（肌肉缺陷、溶酶体贮积病、退行性疾病等）都是由高尔基体功能缺陷引起的。在高尔基体中，碳水化合物部分有序地添加到生物合成货物中是由按顺序起作用的糖基转移酶完成的，因此，早期作用酶的产物是后期作用酶的底物。酶在高尔基体中的位置与糖基化反应相似；早期作用的糖基转移酶富集于顺式/内侧高尔基区室，而后期作用的酶富集于内侧/跨式区室。如何区分分泌货物和高尔基居民，以便货物通过高尔基体顺行移动而保留高尔基居民？利用出芽酵母（Saccharomyces cerevisiae）研究高尔基体中的分选反应，我们发现细胞质蛋白Vps74直接识别高尔基体甘露糖基转移酶亚群的细胞质部分，并需要将其保留在高尔基体中。我们假设Vps74将高尔基体居民分类为逆行通路，然而，文献报道Vps74的人类同源物GOLPH3促进高尔基体的顺行分泌运输。初步数据显示，GOLPH3和Vps74向高尔基膜细胞质小叶的募集需要PtdIns4P的持续合成，PtdIns4P是一种富含高尔基膜的磷酸肌肽，是逆行和逆行高尔基转运所必需的。通过x射线晶体学和脂质结合实验，我们已经确定了GOLPH3和Vps74上的候选PtdIns4P结合位点，并将阐明GOLPH3/Vps74与PtdIns4P复合物的结构。初步数据还显示，在酵母vps74突变体中，PtdIns4P代谢发生改变，这使我们假设vps74和GOLPH3调节4-磷酸化磷酸肌苷的产生和/或周转。高尔基体磷酸肌苷信号的改变被认为是Vps74和GOLPH3功能缺失所导致的分选缺陷的基础。GOLPH3最近被确定为一种候选癌基因，当过表达时导致转化。通过结合酵母和培养的人类细胞的功能研究以及Vps74和GOLPH3的生化、生物物理和结构分析，这些研究将解决PtdIns4P调控在高尔基体中的基本作用，并阐明GOLPH3的功能，从而揭示其在正常和疾病状态中的作用。

DESCRIPTION (provided by applicant): The Golgi apparatus has two primary functions: biosynthesis of glycoproteins and glycolipids, and sorting. These functions underlie the elemental architecture of eukaryotic cells, so understanding how the Golgi functions is of fundamentally important. In addition, many human diseases (muscular deficiencies, lysosomal storage diseases, degenerative disease, etc) result from deficiencies of Golgi function. The ordered addition of carbohydrate moieties to biosynthetic cargo in the Golgi is carried out by glycosyltransferases that function sequentially, such that the products of early acting enzymes are substrates for later-acting enzymes. The location of enzymes in the Golgi stack parallels the glycosylation reactions; early- acting glycosyltransferases are enriched in cis/medial Golgi compartments while later-acting enzymes are enriched in medial/trans compartments. How is secretory cargo distinguished from Golgi residents so that cargo moves anterograde through the Golgi while Golgi residents are retained? Using budding yeast (Saccharomyces cerevisiae) to investigate sorting reactions in the Golgi, we discovered that a cytosolic protein, Vps74, directly recognizes the cytosolic portions of a subset of Golgi mannosyltransferases and is required to retain them in the Golgi. We hypothesize that Vps74 sorts Golgi residents into the retrograde pathway, however, the human ortholog of Vps74, GOLPH3, is reported in the literature to promote anterograde secretory transport from the Golgi. Preliminary data show that recruitment of GOLPH3 and Vps74 to the cytosolic leaflets of Golgi membranes requires ongoing synthesis of PtdIns4P, a phosphoinositide that is enriched in Golgi membranes and is required for both anterograde and retrograde Golgi trafficking. Using X-ray crystallography and lipid binding assays, we have identified a candidate PtdIns4P binding site on GOLPH3 and Vps74 and will elucidate a structure of GOLPH3/Vps74 in complex with PtdIns4P. Preliminary data also show that in a yeast vps74 mutant, PtdIns4P metabolism is altered, leading us to hypothesize that Vps74 and GOLPH3 regulate the production and/or turnover of 4-phosphorylated phosphoinositides. Altered phosphoinositide signaling at the Golgi is postulated to underlie the sorting defects that result from a loss of Vps74 and GOLPH3 function. GOLPH3 has recently been identified as a candidate oncogene that results in transformation when overexpressed. By combining functional studies in yeast and cultured human cells with biochemical, biophysical, and structural analyses of Vps74 and GOLPH3, these studies will resolve fundamental roles of PtdIns4P regulation in the Golgi, and elucidate the function of GOLPH3, which will shed light on its role in both normal and disease states. PUBLIC HEALTH RELEVANCE: In order for secreted proteins (hormones, nutrient and ion transporters, signaling receptors, etc) and lipids to function properly, they must be sequentially modified in the Golgi apparatus by enzymes that attach chains of sugar molecules to them. This research project investigates the structure and functions of proteins that function to assemble and maintain the organization of Golgi enzymes. One of these proteins, GOLPH3, has been implicated in causing a wide variety of cancers, so knowledge gained from this project will provide basic information regarding the assembly of the Golgi apparatus and help elucidate how GOLPH3 causes cancer.