The long term objectives are to establish the precise mechanism by which a large number of secific proteins are translocated across or asymmetrically integrated into specific cellular membranes.
The specific aims are to identify and to characterize components of cellular translocaton system which are involved in i) translocation of secretory and lysosomal proteins across the rough endoplasmic reticulum membrane 11) translocation of peri-plasmic proteins across the prokaryotic plasma membrane ii) translocaton of cytoplasmically synthesized proteins into the mitochondrial matrix across the outer and inner mitochondrial membrane iv) translocation of cytoplsmically synthesized proteins into the chloroplast stroma, across the outer and inner chloroplast membrane. Another specific aim is to characterize the """"""""topogenic"""""""" sequences that yeidl asymmetric integration of proteins into membranes using bovine opsin, the rabbit immunoglobulin A receptyor and yeast cytochrome c peroxidase as model system. Translocation and integration can be faithfully reproduced in reconstituted systems whereby the proteins of interest are synthesized de novo in a cell-free translaton system supplemented with the appropriate translocation-competent membranes and organelles. The components of each of the four translocation systems will b eidentified, isolated and characterized. Among the major approaches taken toward this goal are 1) conventional cell-and biochemical fractionation methods to isolate soluble and membrane-associated components for which there is an assay; 2) crosslinking with a recently synthesized 125I labeled and photoactivatable crosslinker to establish nearest neighbor relationships and to identify additional required components; 3) synthetic peptides which may serve as competitive subtrates for receptors and which could then be used for their isolation by affinity chromatography; systhetic peptides will also be used to raise domain-specific antibodies to study function and topology; 4) recombinant DNA technology to clone genes of identified components and to establish their DNA sequence as well as to alter topogenic sequences specifying protein integration into membranes 5) genetic suppression/complementation analysis in yeast to identify additional components of the rough endoplasmic reticulum translocation system.
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