The research described in this proposal is directed towards the identification and biochemical characterization of the protein components involved in translocation of secretory proteins and lysosomal proteins across the rough endoplasmic reticulum membrane. Two components of the translocation apparatus have been isolated a soluble ribonucleoprotein complex termed the signal recognition particle (SRP) and an integral membrane protein termed the signal recognition particle receptor or docking protein. During the proposed funding period, the research will concentrate upon i) further characterization of the role of the SRP receptor during secretory protein translocation, ii) obtaining a more detailed understanding of the mechanism of ribosome binding to the endoplasmic reticulum and iii) experimentally examining the process of nascent chain transport across the membrane. Specifically, monoclonal antibodies which recognize SRP receptor structural domains will be used in combination with in vitro assays of SRP receptor function to examine the interaction of the SRP and other potential components of the translocation apparatus. The role of the SRP receptor during the formation of a functional ribosome-membrane junction will be examined in detail to determine whether in combination with an SRP-arrested ribosome, the SRP receptor is both necessary and sufficient for promoting ribosome attachment. Reconstitution of the SRP receptor into plospholipid vesicles will provide a model system to address this question using in vitro assays developed to monitor ribosome binding. Ribophorins I and II will be isolated under non-denaturing conditions and examined as potential ribosome receptors using specific in vitro assays developed for the detection of proteins which function during ribosome binding. Affinity chromatography reagents will be developed for the isolation of additional proteins which function during translocation. The long term goals of this investigation will be a complete description of the components required for translocation, and the functional reconstitution of all necessary components into a defined phospholipid vesicle system. The mechanism of nascent chain transport across the phospholipid bilayer can then be examined in detail.
Showing the most recent 10 out of 32 publications
