Mechanism(s) of Protein Retention in Mammalian Golgi Apparatus

高尔基体是真核细胞内主要的亚细胞细胞器系统，存在于高尔基体片段内的蛋白质可以被认为是常驻蛋白质，即大部分生命周期都在细胞器中度过的蛋白质，或者是瞬时蛋白质，即在细胞器中加工的蛋白质，但其最终“国籍”是在细胞内或细胞外环境的其他地方。基于先前的研究结果，可以提出三种假设来解释居留性：(1)由于居留性蛋白与瞬时蛋白的移动性受到限制，因此进入运输囊泡的受限，这导致瞬时蛋白优先包含在从高尔基体出芽的非选择性散装运输囊泡中；(2)瞬时蛋白和驻留蛋白在高尔基转运囊泡中的优先捕获，这两类蛋白的迁移率相似；或者(3)在高尔基转运囊泡中等量地包含瞬时蛋白和驻留蛋白，在每一轮转运后，驻留蛋白被回收。当然，这些假设的各种组合是可能的。为了验证这些假设的有效性，将通过光漂白后共聚焦荧光恢复（cFRAP）直接测量两种膜蛋白的迁移率，一种是瞬时高尔基蛋白（水泡性口炎病毒G蛋白），另一种是常驻高尔基蛋白（小鼠半乳糖转移酶）。重组DNA技术将用于在培养的猴细胞中表达特异性半乳糖转移酶。cFRAP实验将在德国海德堡的欧洲分子生物学实验室与Thomas Kreis博士合作进行，使用EMBL设备。这些实验的准备工作将在弗吉尼亚州布莱克斯堡的弗吉尼亚理工学院和州立大学完成。在第二组实验中，将评估分离的高尔基囊泡中瞬时和常驻高尔基蛋白相对于分离的高尔基复合物的相对富集程度。这些实验将完全在VPI-SU进行。这些实验集中在细胞生物学中的一个重要问题上，即，特定的蛋白质如何在区隔细胞中到达它们功能合适的目的地？这些创新实验的结果有望促进我们对这些关键细胞过程的理解。

Proteins present within segments of the Golgi apparatus, a major subcellular organelle system within eukaryotic cells, can be considered to be either resident proteins, i.e., proteins most of whose lifetimes are spent in the organelle, or transient proteins, i.e., proteins which are processed in the organelle but whose ultimate "citizenship" is elsewhere within the cell or extracellular milieu. Based on previous results, three hypotheses can be advanced to explain residency: (1) limited entry into transport vesicles, due to restricted mobility of resident versus transient proteins, which results in the preferential inclusion of transient protein in non-selective, bulk transport vesicles budding from the Golgi apparatus; (2) preferential trapping of transient versus resident proteins in Golgi transport vesicles with the mobility of both protein classes being similar; or (3) equal inclusion of both transient and resident proteins in Golgi transport vesicles with the resident proteins then being retrieved after each round of transport. Various combinations of these hypotheses are, of course, possible. To test the validity of these hypotheses, the mobility of two membrane proteins, a transient Golgi protein, the vesicular stomatitis virus G protein, and a resident Golgi protein, a murine galactosyl transferase, will be measured directly by confocal fluorescent recovery after photobleaching (cFRAP). Recombinant DNA techniques will be used to express the specific galactosyl transferase in cultured monkey cells. The cFRAP experiments will be done in collaboration with Dr. Thomas Kreis, at the European Molecular Biology Laboratory in Heidelberg, Germany, using EMBL facilities. Preparative work for these experiments will be done at Virginia Polytechnic Institute and State University in Blacksburg, VA. In a second set of experiments, the relative enrichment of the transient and resident Golgi proteins in isolated Golgi vesicles relative to isolated Golgi complexes will be assessed. These experiments will be done entirely at VPI-SU. These experiments focus on an important problem in cell biology, namely, how do specific proteins get to their functionally appropriate destinations within the compartmentalized cell? The results of these innovative experiments are expected to advance our understanding of these critical cellular processes.