STRUCTURE/FUNCTION OF GTC, A NOVEL GOLGI PROTEIN COMPLEX

这项工作的总体目标是通过真核细胞的分泌途径进一步了解蛋白质的运输。这一过程对细胞间隔的产生和维持至关重要，因此对细胞的生存能力也是至关重要的。高尔基体运输复合体(GTC)是一种新型的异寡聚体蛋白复合体，最近被鉴定和纯化，其基础是它能在体外促进高尔基体转运。克隆了其中一个亚基的cDNA3个亚基，并用于制备抗体的重组蛋白。亚细胞分离和间接免疫荧光研究表明，GTC定位于高尔基体。这项工作的具体目的是为了阐明GTC在分泌途径中的作用。为了做到这一点，将开发一种高产率的GTC纯化方法，使用免疫印迹法而不是GTC活性作为检测方法。为了克隆每个多肽的cDNA，将从亚基中获得多肽序列数据。重组的GTC亚基将被用来产生多克隆和单克隆的抗GTC抗体，用于结构和功能分析。结构研究将包括GTC亚单位化学计量的严格测定，通过深蚀电子显微镜进行形态检查，在复合体中定位亚单位，尝试产生功能亚复合体，以及通过化学交联和可能的双杂交研究进行最近邻分析。GTC的细胞内定位将使用亚细胞分离、共聚焦和免疫金显微镜进行详细分析。GTC功能将在体内、高表达或低表达的细胞中进行分析，以确定GTC功能处于早期分泌途径的哪个阶段。这些结果将通过使用监测几个早期运输步骤的半完整细胞系统来证实。为了确定GTC是否影响囊泡萌发或膜对接/融合，将检查两种体外试验中潜在的GTC要求；一种仅重构囊泡萌发，另一种仅测量膜对接/融合。最后，由于GTC与高尔基体膜密切相关，因此将进行鉴定和纯化GTC受体的实验。这些研究将首次深入了解这种新的蛋白质复合体在细胞内蛋白质运输过程中的作用。

The overall goal of the work proposed here is to further understanding of protein traffic through the secretory pathway of the eucaryotic cell. This process is crucial for the generation and maintenance of cellular compartment, and hence for viability. GTC (for Golgi Transport Complex) is a novel hetero-oligomeric protein complex that has recently been identified and purified based on its ability to facilitate transport through the Golgi in vitro. A cDNA fore one of the subunits was cloned, and used to generate recombinant protein for antibody production. Use of the antibody in subcellular fractionation and indirect immunofluorescence studies showed that GTC is localized to the Golgi. The specific aims of the work proposed here are designed to elucidate the role of GTC in the secretory pathway. To do this, a high yield GTC purification will be developed employing immunoblotting, rather than GTC activity, as a detection method. Peptide sequence data will be obtained from the subunits in order to clone cDNAs for each of the polypeptides. Recombinant GTC-subunits will be used to generate polyclonal and monoclonal anti-GTC antibodies for structural and functional analyses. The structural studies will entail a rigorous determination of GTC subunit stoichiometry, morphological examination by deep-etch electron microscopy, localization of the subunits within the complex, an attempt to generate functional subcomplexes, and nearest neighbor analyses by chemical cross-linking and perhaps two-hybrid studies. The intracellular localization of GTC will be analyzed in detail using subcellular fractionation, and confocal and immuno-gold microscopy. Potential residence within the Golgi-matrix or on COPI-coated vesicles will be examined biochemically GTC function will be analyzed in vivo, in cells over-expressing or under-expressing GTC subunits to determine at what stage of the early secretory pathway GTC functions. These results will be corroborated through use of semi-intact cell systems that monitor several early transport steps. In an effort to determine whether GTC affects vesicle budding, or membrane docking/fusion the potential GTC requirement in two in vitro assays will be examined; one reconstitutes only vesicle budding, and the other measures only membrane docking/fusion. Lastly, since GTC is tightly associated with the Golgi membrane, experiments to identify and purify a putative GTC receptor will be undertaken. These studies will provide the first insights into the role of this novel protein complex in the process of intracellular protein traffic.