Genetic Analysis of Membrane Traffic in Mammalian Cells

95-13244 Draper真核细胞由不同的膜内结构组成，每个内膜结构都有独特的蛋白质和脂类补充。尽管动态膜运输涉及将膜组分从一个隔室转移到另一个隔室，但膜保持其独特的组成。这种动态的膜运输被认为是通过起源于供体膜、迁移到受体膜并融合到受体隔室的膜囊来实现的。小泡形成的机制以及小泡识别和融合到受体隔室的机制还知之甚少。这项提议涉及到对膜贩运中的分子事件的洞察。德雷珀博士分离出了CHO细胞的温度敏感型突变体，这些突变体由五个互补基团组成，即End1到End5，它们在膜运输的某些方面存在缺陷。这项建议的重点是深入了解End4互补基团的突变体的性质，并分离新的突变体。End4组的突变体在内质网到高尔基体运输的不允许温度处存在障碍，这与后者的耗散有关。该建议的目的是建立一个体外系统，对End4突变体进行生化分析，通过互补克隆受影响的基因，并分离和鉴定对蛋白质毒素具有抗性且膜转运有缺陷的突变体。真核细胞由不同的膜内结构组成，每个内膜结构都有蛋白质和脂类的独特补充。尽管动态膜运输涉及将膜组分从一个隔室转移到另一个隔室，但隔室保持其独特的膜成分。这种动态的膜运输被认为是通过在供体隔室形成膜囊泡，这些膜囊泡迁移到受体隔室，并融合到受体隔室来实现的。小泡形成的机制，即小泡识别和融合到受体隔室的机制还不是很清楚。这项提议涉及到对膜贩运的分子事件的洞察。***

95-13244 Draper Eukaryotic cells consist of various endomembranous structures, each with a unique complement of proteins and lipids. Despite a dynamic membrane traffic involving transfer of membrane components from one compartment to another, the membranes maintain their distinct composition. This dynamic membrane traffic is presumed to occur by membrane vesicles that originate at donor membranes, migrate to acceptor membranes, and fuse to the acceptor compartment. The mechanisms by which vesicles are formed and by which vesicles identify and fuse to the acceptor compartment are poorly understood. This proposal is concerned with gaining insights into the molecular events in membrane trafficking. Dr. Draper has isolated temperature-sensitive mutants of CHO cells that comprise five complementation groups, End1 through End5, that are defective in certain aspects of membrane trafficking. The focus of this proposal is to gain insights into the nature of the mutants of the End4 complementation group and to isolate new mutants. Mutants of the End4 group have a block at the non-permissive temperature in endoplasmic reticulum to Golgi transport that correlates with a dissipation of the latter. The objectives of the proposal are to establish an in vitro system to biochemically analyze the End4 mutants, to clone the affected gene by complementation, and to isolate and characterize mutants that are resistant to protein toxins and are also defective in membrane trafficking. %%% Eukaryotic cells consist of various endomembranous structures, each with a unique complementation of proteins and lipids. Despite a dynamic membrane traffic involving transfer of membrane components from one compartment to another, the compartments maintain their distinct membrane composition. This dynamic membrane traffic is presumed to occur by the formation of membrane vesicles at the donor compartment, migration of these to the acceptor compartment, and fusion to the acceptor compartment. The mechanisms by which vesicles are formed, by which vesicles identify and fuse to the acceptor compartment are poorly understood. This proposal is concerned with gaining insights into the molecular events of membrane trafficking. ***