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

描述(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和分选。这些功能构成了真核细胞的基本结构，因此了解高尔基体的功能是至关重要的。此外，人类的许多疾病(肌肉缺陷、溶酶体储存疾病、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糖基转移酶按顺序起作用的，因此早期作用的酶的产物是后来作用的酶的底物。酶在高尔基体堆叠中的位置与糖基化反应平行；早期作用的糖基转移酶在顺式/内侧高尔基体室中富含，而晚期作用的酶在中间/反式间隔室中富含。如何区分秘密货物和高尔基居民，使货物顺行通过高尔基群岛，同时保留高尔基居民？利用发芽酵母(Saccharmyces Cerevisiae)研究高尔基体中的分选反应，我们发现一种胞浆蛋白Vps74直接识别高尔基体甘露糖基转移酶亚集的胞液部分，并需要将它们保留在高尔基体中。我们假设Vps74将高尔基体居民分类为逆行途径，然而，文献报道Vps74的人类同源基因GOLPH3促进高尔基体的顺行分泌运输。初步数据显示，GOLPH3和Vps74在高尔基体膜的胞浆小叶上的募集需要持续合成PtdIns4P，这是一种富含在高尔基体膜上的磷脂酰肌醇，是顺行和逆行高尔基体运输所必需的。利用X射线结晶学和脂质结合分析，我们已经确定了GOLPH3和Vps74上PtdIns4P的候选结合部位，并将阐明GOLPH3/Vps74与PtdIns4P形成的络合物的结构。初步数据还显示，在酵母vps74突变体中，PtdIns4P的代谢发生了变化，这导致我们假设Vps74和GOLPH3调节4-磷酸化肌醇的生产和/或周转。高尔基体上肌醇磷脂信号的改变被认为是Vps74和GOLPH3功能丧失导致的分类缺陷的基础。GOLPH3最近被确定为一种候选癌基因，当过度表达时会导致转化。通过在酵母和培养的人类细胞中的功能研究与Vps74和GOLPH3的生化、生物物理和结构分析相结合，这些研究将解决PtdIns4P调控在高尔基体中的基础作用，并阐明GOLPH3的功能，这将有助于揭示其在正常和疾病状态下的作用。 与公共健康相关：为了使分泌的蛋白质(激素、营养和离子转运体、信号受体等)和脂类正常工作，它们必须在高尔基体中被将糖分子链连接到它们上的酶顺序修饰。这项研究项目调查了蛋白质的结构和功能，这些蛋白质的功能是组装和维持高尔基体酶的组织。其中一种名为GOLPH3的蛋白质与多种癌症有关，因此从该项目中获得的知识将提供有关高尔基体组装的基本信息，并有助于阐明GOLPH3如何致癌。

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.