This is a proposal to characterize the structure and function, as well as regulation of expression, of three rought endoplasmic reticulum (RER)-specific membrane proteins, ribophorin I, II and the 83 Kd protein. These proteins are thought to be part of the apparatus that translocates and processes polypeptides synthesized in membrane-bound polysomes. Procedures will be developed to reconstitute rough microsomal membranes from solubilized components and the functional capacity of these vesicles with respect to ribosome binding, signal peptide cleavage, N-glycosylation and translocation of secretory and membrane proteins will be assessed. The effects of monoclonal and polyclonal antibodies against the aforementioned RER membrane proteins on the various functional capacities of the reconstituted rough microsomes (RM) will provide insights into the specific function of each protein in the translocation process. The amino acid sequences of the proteins will be deduced from the corresponding cDNA clones, and the spatial dispositions of the polypeptides with respect to the membrane will be determined using immunocytochemical and chemical labeling approaches and will be compared with those predicted from the sequences. We will also investigate near neighbor relationships amongst the components of the translocation apparatus using heterobifunction cross-linking reagents. By disrupting the genes corresponding to the ribophorins or the 83 Kd protein in yeast cells, or by using anti-sense RNA inhibition of gene expression on cells in which the development of the RER can be induced, the importance of these proteins for translocation and processing of products made in membrane bound polysomes will be assessed. In vitro transcription and translation of mutated or of chimeric genes for these three proteins will be used to define insertion and stop transfer signals, while transfection experiments will serve to identify domain(s) in the polypeptides that lead to their retention in the rough domain of the ER. Analysis of the structure of the genes for these membrane proteins should ultimately provide information on the regulatory elements involved in the coordinate expression of the components of the translocation apparatus.
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