STRUCTURE/FUNCTION OF GTC, A NOVEL GOLGI PROTEIN COMPLEX

本文提出的总体目标是进一步了解真核细胞分泌途径中的蛋白质运输。这个过程对细胞区室的产生和维持至关重要，因此对细胞的生存能力至关重要。高尔基转运复合物（GTC）是一种新型的异聚寡聚蛋白复合物，最近被鉴定和纯化，基于其在体外促进通过高尔基体转运的能力。克隆了其中一个亚基的cDNA，并利用其生成重组蛋白来生产抗体。利用该抗体进行亚细胞分离和间接免疫荧光研究表明，GTC定位于高尔基体。这里提出的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，将开发一种高产的GTC纯化方法，采用免疫印迹法，而不是GTC活性作为检测方法。为了克隆每个多肽的cdna，将从亚基中获得肽序列数据。重组gtc亚基将用于生成多克隆和单克隆抗gtc抗体，用于结构和功能分析。结构研究将需要严格确定GTC亚基化学计量学，通过深蚀刻电子显微镜进行形态学检查，亚基在复合物内的定位，尝试产生功能亚复合物，以及通过化学交联和可能的双杂交研究进行最近邻分析。我们将使用亚细胞分离、共聚焦和免疫金显微镜详细分析GTC的细胞内定位。GTC在高尔基基质或copi包被囊泡内的潜在驻留将被生化检测。GTC的功能将在体内，在过表达或低表达GTC亚基的细胞中进行分析，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运输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，我们将检查两种体外实验中潜在的GTC需求；一个只重建囊泡出芽，另一个只测量膜对接/融合。最后，由于GTC与高尔基膜紧密相关，因此将进行鉴定和纯化假定的GTC受体的实验。这些研究将首次深入了解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。

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.