CLONING AND ANALYSIS OF TETRAHYMENA SOMATIC CHROMOSOMES

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This proposal seeks to identify and characterize the cellular machinery that governs the intracellular transport of plasma membrane, and secretory proteins from their site of synthesis on the rough endoplasmic reticulum (ER) to their site of function. The inability of a cell to direct proteins along the correct intracallular pathway may lead to serious physiological problems. For example, patients that suffer from a number of genetic diseases synthesize biologically active proteins, but fail to deliver them to their site of function. Two unique probes will be used to examine different steps in transport. The first probe is a hybrid protein, GHHA, encoded by a fusion of the cDNA's for rat growth hormone and a fragment of influenza hemagglutinin. GHHA accumulates in a region of the ER tentatively identified as a pre-Golgi subcompartment. This subcompartment will be characterized, in situ and as isolated microsomes, to determine if it is an essential ER subcompartment with unique functions that distinguish it from the rough ER. The effect of GHHA on the transport of other proteins to the Golgi will be assessed by immunofluorescence and by measuring the rate at which these proteins are modified by Golgi enzymes. The relative abundance of various proteins and enzyme activities in the rough ER and pre-Golgi will be determined. The second probe is a hybrid protein, GHG3, encoded by a fusion of the cDNA's for rat growth hormone and a fragment of vesicular stomatitis virus G protein. GHG3 is transported rapidly to lysosomes and degraded. GHG3 will be used to examine mechanisms of transport to the cell surface and lysosomes, by determining whether it contains three distinct sorting signals, or whether it is transported by a previously unrecognized autophagic pathway that directs misfolded proteins to the lysosome for degradation. The role of different subdomains of GHG3 on transport will be determined by using site-directed mutagenesis. The structure of these mutant proteins will be determined by limited proteolysis and measuring binding to growth hormone receptor in order to relate structure of the mutant proteins to their transport properties.