Mechanism(s) of Protein Retention in Mammalian Golgi Apparatus

哺乳动物高尔基体中蛋白质保留的机制

• 批准号: 9022817
• 负责人: Brian Storrie
• 金额: $ 6.9万
• 依托单位: Virginia Polytechnic Institute and State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9022817&HistoricalAwards=false
• 关键词: Mechanism; Protein; Retention; Mammalian; Golgi

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.

存在于高尔基体片段内的蛋白质，是一种主要的 真核细胞内的亚细胞器系统，可以是 被认为是常驻蛋白质，即，大多数蛋白质 它们的生命都是在细胞器中度过的，或者说是短暂的蛋白质， 也就是说，在细胞器中加工但其 最终的“公民身份”是在细胞内的其他地方， 细胞外环境 根据先前的研究结果，三个假设 可以提前解释居留权：（1）有限进入 运输囊泡，由于限制流动性的居民与 瞬时蛋白，这导致优先包含 非选择性、大量运输囊泡中的瞬时蛋白质 从高尔基体;（2）优先捕获的瞬态 与高尔基体运输囊泡中的常驻蛋白质相比， 两种蛋白质类的迁移率相似;或（3）相等 在高尔基体中包含瞬时蛋白和驻留蛋白 携带蛋白质的运输囊泡 在每一轮运输之后。 这些的各种组合 当然，假设是可能的。 为了测试这些的有效性， 假设，两种膜蛋白的流动性，一种瞬时的 高尔基体蛋白，水泡性口炎病毒G蛋白，和 常驻高尔基体蛋白，一种鼠半乳糖基转移酶， 直接通过共聚焦荧光恢复测量， 光漂白（cFRAP）。 将使用重组DNA技术 在培养猴中表达特异性半乳糖基转移酶 细胞 cFRAP实验将与以下机构合作完成 年欧洲分子生物学实验室的托马斯克瑞斯博士说， 海德堡，德国，使用EMBL设施。 为...... 这些实验将在弗吉尼亚理工学院进行 和弗吉尼亚州布莱克斯堡的州立大学。 的第二集合中 实验中，瞬态和驻留的相对富集 分离的高尔基体囊泡中的高尔基体蛋白相对于分离的 将评估高尔基复合体。 这些实验将在 在VPI-SU。 这些实验聚焦于细胞生物学中的一个重要问题， 也就是说，特定的蛋白质是如何发挥其功能的， 合适的目的地吗 的 这些创新实验的结果有望推动我们的 了解这些关键的细胞过程。