Cloned endoplasmic reticulum membrane glycoprotein genes will be used to probe the primary sequence requirements directing the sorting of glycoproteins into specific membrane compartments. Standard techniques of molecular biology will be employed for manipulation of genetic material which will be expressed in a variety of eukaryotic expression vectors. We are specifically interested in endoplasmic reticulum (ER) glycoproteins because defined alteration in their genes resulting in expressed products moving from the ER to other compartments, such as the plasma membrane, can be positively interpreted as to the nature of the signal directing the sorting behavior. We will use rotavirus as a model system, since it buds into the RER, where glycoproteins it encodes are specifically located. Our studies with this system to date have produced novel and important results which identify a primary sequence responsible for anchoring a membrane glycoprotein in the ER and lead to the implication that secreted forms of it probably do not require a receptor to leave the ER. We plan to thoroughly characterize the primary sequence requirements for the ER membrane anchor by gene manipulation and in vitro mutagenesis techniques. In conjunction, we wish to characterize asparagine-linked oligosaccharides from glycoproteins in specific elements of the ER early in the secretory or exocytosis pathway to elucidate the extent of the mannosidase processing. We will analyze the structure of certain oligosaccharide intermediates by methods which include 500 MHz 1H-NMR spectroscopy. We plan to relate the amount of processing on yeast invertase carbohydrate in selected early secretory pathway mutant complementation groups which show evidence of carbohydrate differences. A precise analysis of the extent of carbohydrate processing particular compartments should provide useful markers for the compartments, and may help to define new ones. Detailed analysis of processing of the rotavirus VP7 has implicated a sub-compartment of the ER in animal cells in which this glycoprotein is processed and we plan to further characterize this compartment.
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