proteins are targeted to and maintained within specific subcellular sites. Knowle of these targeting mechanisms will further our understanding of the exquisitely precise architecture of the cell and of abnormalities of subcellular structures which occur in many diseases. IN particular, the long-term objective of this research is to understand the mechanisms responsible for the retention of the proteins glucuronidase, the esterase egasyn and other carboxyl esterases within the lumen of the endoplasmic reticulum (ER) of hepatocytes and other cells. The esterases are important in detoxification of xenobiotics, and ER glucuronidase modulates levels of bilirubin glucuronides. Egasyn and glucuronidase, which exist as a complex, serve as a model system which may illustrate general mechanisms by which lumenal proteins are maintained within the ER.
The specific aims are to: 1) Determine the structural features of egasyn which are responsible for its retention within the ER; 2) Determine the strilctural features of other esterases of the ER and plasma which are responsible for their retention in or secretion from the ER and to compare these features with corresponding signals on other lumenal or secreted proteins; 3) To identify and partially characterize the receptor which retains egasyn and other carboxyl esterases within the ER and, 4) Test if the serpin-like sequence regions of the glucuronidase propeptide function in retention of glucuronidase within the ER. These experiments will utilize in vitro mutagenesis and expression of cloned egasyn and esterase cDNAs and of fusion genes containing specific regions of the cloned egasyn and glucuronidase cDNAs. Cloned cDNAs of other esterases of the ER lumen will be prepared and compared so that structural features responsible for their retention within the ER can be similarly investigated. Expression systems will include several cultured cell lines. When appropriate, these techniques will be supplemented by the use of synthetic peptide reagents corresponding to defined regions of the above proteins. Affinity chromatography techniques and/or antiidiotype approaches will be used to identify and partially characterize the receptor(s) which recogn'lzes the ER retention signal on esterases.
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