Eukaryotic cells have mechanisms to recognize, sort, and transport transmembrane proteins between organelles of the exocytic and endocytic pathways. With few exceptions, little is known of the features of individual proteins that are recognized during this process. In the application experiments are proposed to: (1) locate the features of two chimeric glycoproteins, HAG and HAgC, that are responsible for directing these proteins into the endocytic pathway; (2) examine the mechanism by which HAgC is targeted to lysosomes whereas HAG is not; (3) determine whether the feature of HAG and HAgC that specifies their internalization also allows them to bind to components of clathrin coats; (4) examine the influence of foreign transmembrane and cytoplasmic domains on the structure of the HA external domain; (5) determine whether the HA transmembrane and cytoplasmic domains influence the efficiency with which HA is correctly sorted to the apical surface of polarized epithelial cells. A series of chimeric genes will be constructed using oligonucleotide directed mutagenesis. These genes will be expressed from SV40 and bovine papilloma virus vectors. The proteins encoded by these genes will be analysed for (i) rate of transport to the cell surface; (ii) rate of internalization; (iii) change in shape, stability, and function of the HA external domain. Certain of these genes will be expressed in polarized epithelial cells and sorting of altered HA proteins will be investigated. These experiments will contribute towards an understanding of the molecular mechanisms controling transport and sorting of cell surface proteins.
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