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

描述（由申请人提供）：高尔基体具有两个主要功能：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）由高尔基体功能缺陷引起。 有序添加碳水化合物部分到高尔基体中的生物合成货物是通过糖基转移酶进行的，糖基转移酶顺序发挥作用，使得早期作用酶的产物是后期作用酶的底物。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/内侧高尔基体隔室中富集，而晚期作用的酶在内侧/反式隔室中富集。分泌性货物如何与高尔基体居民区分，从而货物通过高尔基体顺行而高尔基体居民被保留？使用芽殖酵母（酿酒酵母），以调查在高尔基体中的分选反应，我们发现，一个胞质蛋白，Vps 74，直接识别高尔基甘露糖基转移酶的一个子集的胞质部分，并需要保留在高尔基体。我们假设Vps 74将高尔基体居民分为逆行通路，然而，文献中报道了Vps 74的人类直系同源物GOLPH 3促进高尔基体的顺行分泌运输。初步数据显示，募集GOLPH 3和Vps 74到高尔基体膜的胞质小叶需要持续合成PtdIns 4P，PtdIns 4P是一种富含高尔基体膜的磷酸肌醇，并且是顺行和逆行高尔基体运输所需的。使用X射线晶体学和脂质结合分析，我们已经确定了一个候选PtdIns 4P的GOLPH 3和Vps 74的结合位点，并将阐明与PtdIns 4P复合的GOLPH 3/Vps 74的结构。初步数据还表明，在酵母vps 74突变体中，PtdIns 4P代谢被改变，导致我们假设Vps 74和GOLPH 3调节4-磷酸化磷酸肌醇的产生和/或周转。在高尔基体改变的磷酸肌醇信号被假定为基础的分拣缺陷，导致从Vps 74和GOLPH 3功能的损失。GOLPH 3最近被鉴定为当过表达时导致转化的候选癌基因。通过将酵母和培养的人类细胞中的功能研究与Vps 74和GOLPH 3的生物化学、生物物理学和结构分析相结合，这些研究将解决PtdIns 4P调节在高尔基体中的基本作用，并阐明GOLPH 3的功能，这将阐明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.