STRUCTURE/FUNCTION OF GTC, A NOVEL GOLGI PROTEIN COMPLEX

本文的总体目标是进一步了解真核细胞分泌途径中蛋白质的运输。这一过程对于细胞区室的产生和维持至关重要，因此对于生存力至关重要。高尔基体转运复合物（Golgi Transport Complex，GTC）是一种新型的异源寡聚蛋白复合物，最近被鉴定和纯化，其基础是其在体外促进通过高尔基体转运的能力。克隆一个亚基前的cDNA，并用于产生用于抗体生产的重组蛋白。使用的抗体在亚细胞分级分离和间接免疫荧光研究表明，GTC定位于高尔基体。这里提出的工作的具体目标是阐明GTC在分泌途径中的作用。为此，将采用免疫印迹法（而非GTC活性）作为检测方法开发高产率GTC纯化。将从亚基获得肽序列数据，以便克隆每种多肽的cDNA。重组GTC亚基将用于产生多克隆和单克隆抗GTC抗体，用于结构和功能分析。结构的研究将需要严格的确定GTC亚基的化学计量，形态学检查深蚀刻电子显微镜，定位的复杂内的亚基，试图产生功能subcomplex，和最近邻分析化学交联，也许双杂交研究。GTC的细胞内定位将使用亚细胞分级分离、共聚焦和免疫金显微镜进行详细分析。在高尔基体基质内或在COPI包被的囊泡上的潜在驻留将被生物化学地检查，GTC功能将被体内分析，在过表达或低表达GTC亚基的细胞中，以确定GTC在早期分泌途径的哪个阶段起作用。这些结果将通过使用监测几个早期转运步骤的半完整细胞系统得到证实。为了确定GTC是否影响囊泡出芽或膜对接/融合，将检查两种体外试验中的潜在GTC要求;一种仅重建囊泡出芽，另一种仅测量膜对接/融合。最后，由于GTC与高尔基体膜紧密相关，因此将进行鉴定和纯化推定的GTC受体的实验。这些研究将为这种新型蛋白质复合物在细胞内蛋白质运输过程中的作用提供第一个见解。

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.