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

高尔基体中蛋白质分选的 PtdIns 4 激酶调节

基本信息

批准号：
8338792
负责人：
Christopher G Burd
金额：
$ 28.74万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-30 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8338792
关键词：
1-Phosphatidylinositol 4-Kinase Architecture Binding Binding Sites Biochemical Biological Assay Cancer Etiology Carbohydrates Catalytic Domain Cell membrane Cells Coat Protein Complex I Collaborations Complex Conflict (Psychology)Data Defect Degenerative Disorder Destinations Disease Enzymes Equilibrium Eukaryotic Cell Glycolipids Golgi Apparatus Homeostasis Hormones Human Ions Knowledge Light Lipid Binding Lipids Literature Location Lysosomal Storage Diseases Malignant Neoplasms Mannosyltransferases Medial Medial Golgi Membrane Membrane Proteins Metabolism Modification Muscle Nutrient Oncogenes Organelles Orthologous Gene Pathway interactions Peripheral Phosphatidylinositols Phosphoric Monoester Hydrolases Positioning Attribute Process Production Protein Family Proteins Proteomics RNA Interference Reaction Receptor Signaling Regulation Reporting Research Project Grants Resolution Role Saccharomyces cerevisiae Saccharomycetales Signal Transduction Sorting - Cell Movement Structure System Testing Time Vesicle X-Ray Crystallography Yeasts base enzyme substrate genetic analysis glycoprotein biosynthesis glycosylation glycosyltransferase human disease in vivo mutant overexpression protein function research study retrograde transport sugar tool trafficking

项目摘要

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.

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至关重要。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。碳水化合物部分向高尔基体中的生物合成货物的有序添加是由顺序起作用的糖基转移酶进行的，使得早期作用酶的产物是后期作用酶的底物。高尔基体堆栈中酶的位置与糖基化反应平行；早期作用的糖基转移酶富集在顺式/内侧高尔基体区室中，而后期作用的酶则富集在内侧/反式区室中。如何区分分泌货物和高尔基居民，以便货物顺行通过高尔基体，而保留高尔基居民？使用芽殖酵母（酿酒酵母）研究高尔基体中的分选反应，我们发现胞质蛋白 Vps74 直接识别高尔基体甘露糖基转移酶子集的胞质部分，并且需要将它们保留在高尔基体中。我们假设 Vps74 将高尔基体分类为逆行途径，然而，文献报道 Vps74 的人类直系同源物 GOLPH3 可促进高尔基体的顺行分泌运输。初步数据表明，将 GOLPH3 和 Vps74 募集到高尔基体膜的胞质小叶需要持续合成 PtdIns4P，PtdIns4P 是一种富含高尔基体膜的磷酸肌醇，是顺行和逆行高尔基体运输所必需的。使用 X 射线晶体学和脂质结合测定，我们已经确定了 GOLPH3 和 Vps74 上的候选 PtdIns4P 结合位点，并将阐明与 PtdIns4P 复合的 GOLPH3/Vps74 的结构。初步数据还表明，在酵母 vps74 突变体中，PtdIns4P 代谢发生改变，这使我们推测 Vps74 和 GOLPH3 调节 4-磷酸化磷酸肌醇的产生和/或周转。据推测，高尔基体中磷酸肌醇信号的改变是由于 Vps74 和 GOLPH3 功能丧失而导致的分选缺陷的基础。 GOLPH3 最近被确定为候选癌基因，过度表达时会导致转化。通过将酵母和培养的人类细胞的功能研究与 Vps74 和 GOLPH3 的生化、生物物理和结构分析相结合，这些研究将解决高尔基体中 PtdIns4P 调节的基本作用，并阐明 GOLPH3 的功能，从而阐明其作用在正常和疾病状态下。