The goal of this proposal is to identify and characterize the cellular machinery that governs the intracellular transport of plasma membrane, and secretory, proteins. These processes determine the ultimate subcellular location of those polypeptides that are synthesized by ribosomes bound to the endoplasmic reticulum. Related processes guide the movement of proteins, such as membrane receptors, that shuttle between the plasma membrane and internal membrane systems. Alterations in the capacity of a cell to correctly direct a protein has been shown to be the molecular basis of several genetic diseases. Recombinant DNA technology will be used to construct genes encoding altered or novel proteins that will serve as substrate analogs for the intracellular transport mechanism. The well characterized envelope glycoproteins, hemagluginin (HA) of influenza virus and the glycoprotein (G) of vesicular stomatitis virus, will be used as model plasma membrane proteins. Rat growth hormone will be used as a model secretory protein. Regions of these proteins will be tested for their transport properties by recombining cDNA fragments that encode an entire protein subdomain with genetic elements that insure protein expression and synthesis on ribosomes bound to the ER. In addition, hybrid mutants that contain domains of two proteins linked together will be constructed to test the ability of one domain to influence the transport of the other. The recominant genes will be expressed, under the control of the SV40 early promoter, in two related monkey fibroblast cell lines (CV-1 and COS-7) to study transport from the endoplasmic reticulum (ER) to the cell surface. Two mutant proteins which have already been constructed will be studied further: 1) a growth hormone-HA hybrid protein that accumulates in a membrane-bounded compartment, located between the ER and the Golgi apparatus, and 2) a growth hormone-G hybrid protein in which two peptides of G have been identified that may serve as the sorting signals recognized by the transport mechanism. The previously unidentified """"""""pre-Golgi"""""""" compartment in which the growth hormone-HA hybrid protein accumulates will be isolated and characterized.
| Rizzolo, L J (1989) A growth hormone-vesicular stomatitis virus G hybrid protein is rapidly degraded in lysosomes following transport to the cell surface. Eur J Cell Biol 49:92-8 |
| Rizzolo, L J; Kornfeld, R (1988) Post-translational protein modification in the endoplasmic reticulum. Demonstration of fatty acylase and deoxymannojirimycin-sensitive alpha-mannosidase activities. J Biol Chem 263:9520-5 |
