The long-term objectives of the proposed study are a) to determine what information is necessary to sort a completed protein from the cytoplasm and bind it to its correct intracellular membrane, b) to genetically and biochemically define the components of the cell which participate in organelle delivery, and c) to define the protein sequences and membrane bound components which are required for the posttranslational secretion of proteins from one intracellular soluble compartment to another. Since the delivery of many of the cells proteins to different subcellular organelle membranes including mitochondria, the nucleus, endoplasmic reticulum, and vacuoles occurs following their synthesis in the cytoplasm a detailed analysis of the rules and components required for this process is essential to understand the assembly and turnover of intracellular membranes. In order to answer these questions, a yeast model will be utilized to examine the ability of organelle specific protein sequences to deliver a reporter protein which is fused to it to the correct subcellular space. The delivery and import sequences for proteins targeted to the mitochondria will be first determined by systematic deletion and replacement of different regions of F1-ATPase and adenine nucleotide translocator proteins by gene manipulation. Analysis of the function of these sequences in protein localization will utilize the combination of immunological and membrane impermeable enzymatic and chemical probes to localize the protein within the organelle membrane. Since the in vivo mitochondrial delivery of one of the hybrid proteins has been shown to inhibit the normal function of mitochondria and provide an easily scorable phenotype, a genetic approach can be initiated to define the genes required for mitochondrial import. Furthermore, endogenous molecules which bind to targeting sequences will be selected by affinity purification methods exploiting isolated hybrid protein constructs. The combination of biochemical, immunological and genetic studies is proposed to initiate an analysis of the delivery and biogenesis of yeast nuclear membrane pore complex-lamina proteins. This analysis will provide the initial probes to extend the analysis of cellular components and signals responsible for cytoplasmic sorting.
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