The research described in this proposal is directed towards elucidating the mechanism by which proteins are translocated across the rough endoplasmic reticulum. Particular emphasis will be placed on (a) the analysis of GTP-dependent events that occur during early phases of the protein translocation reaction and on (b) the biochemical, molecular and functional characterization of a 34 kD integral membrane protein that can be crosslinked to in vitro assembled translocation intermediates. GTP binding proteins perform a pivotal role at early stages of the protein translocation reaction. Three subunits of the signal recognition particle (SRP) and the SRP receptor are predicted to be GTP-binding proteins. Insight into the precise role of these proteins will be obtained by defining all three GTP hydrolysis cycles with respect to (a) regulatory components that initiate guanine nucleotide exchange, (b) downstream effector proteins or targets and (c) proteins that activate GTP hydrolysis., The GTP hydrolysis cycle of the SRP receptor alpha subunit will be investigated by combining site directed mutagenesis of the protein with in vitro analysis of SRP receptor function. Assays will be developed to determine the significance of the GTP binding sites in the 54 kD subunit of SRP and the beta subunit of the SRP receptor. Reaction intermediates will be trapped by deleting GTP or substituting nonhydrolyzable GTP analogues. GTP binding and hydrolysis assays will be developed to identify the proteins that regulate the GTP hydrolysis cycle of SRP54. The long term goal of this portion of the project is to learn how sequential or interlocking GTP hydrolysis cycles control the selective delivery of ribosomes to me surface of the rough endoplasmic reticulum. Proteins that are proposed to mediate nascent chain transport have been identified in mammalian and yeast systems using distinct experimental approaches. To date, proteins identified in the mammalian transport reaction do not have obvious homologues in the yeast transport reaction. Chemical crosslinking has been used to detect an integral membrane protein (imp34) that is adjacent to polypeptides undergoing transport across mammalian microsomal membranes. Imp-34 will be purified from canine microsomal membranes and a cDNA clone encoding the protein will be sequenced to allow comparison with the yeast Sec6l, Sec62 and Sec63 proteins. More importantly, liposome reconstitution assays will be used to evaluate the role of imp-34 in the protein transport reaction. The long term goal of this project is to understand how proteins are selectively transported across membrane bilayers. Once all of the necessary components have been identified, these protein translocation components can be reconstituted into phospholipid vesicles and the transport process can be analyzed in detail.
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