Mechanism(s) of Protein Retention in Mammalian Golgi Apparatus

高尔基体是真核细胞内的一个主要亚细胞器官系统，高尔基体各节段中存在的蛋白质可以被认为是驻留蛋白质，即其大部分生命周期在细胞器中度过的蛋白质，或瞬时蛋白质，即在细胞器中加工但其最终的“公民身份”在细胞内或细胞外环境中的其他地方的蛋白质。根据已有的结果，可以提出三个假说来解释滞留：(1)由于驻留蛋白相对于瞬时蛋白的迁移率受到限制，从而限制进入运输小泡，这导致瞬时蛋白优先包含在从高尔基体萌发的非选择性的大宗运输囊泡中；(2)暂时性蛋白相对于驻留蛋白优先捕获在高尔基体运输囊泡中，两类蛋白的迁移率相似；或者(3)高尔基体运输囊泡中瞬时蛋白和驻留蛋白的等量包含，驻留蛋白在每一轮运输后被回收。当然，这些假设的各种组合是可能的。为了验证这些假说的有效性，我们将通过光漂白后的共聚焦荧光恢复(CFRAP)直接测量两种膜蛋白，瞬时高尔基蛋白、水泡性口炎病毒G蛋白和常驻高尔基蛋白(鼠半乳糖转移酶)的迁移率。重组DNA技术将用于在培养的猴细胞中表达特异性的半乳糖转移酶。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，使用EMBL设施进行。这些实验的准备工作将在弗吉尼亚理工学院和弗吉尼亚州布莱克斯堡的州立大学进行。在第二组实验中，将评估相对于孤立的高尔基复合体，在分离的高尔基囊泡中暂时性和驻留的高尔基蛋白的相对富集度。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分隔的细胞中到达其功能适当的目的地？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.