The research project is directed towards elucidating the mechanism responsible for translocation of proteins across the rough endoplasmic reticulum (RER). The first specific aim will be to determine how the signal recognition particle (SRP) and the SRP receptor (SR) mediate the efficient high-fidelity delivery of the ribosome-nascent chain (RNC) complex to the translocation channel when the ribosome, a high affinity ligand for the Sec61 core of the translocation channel, is presented in vast molar excess. Competition experiments will indicate whether synergism between the SR and the Sec61 complex accounts for the specificity and affinity for SRP-RNC targeting to the RER. Quantification of membrane bound ribosomes will reveal whether inactive ribosomes that occlude the Sec61 complex are displaces when SRP delivers a RNC complex to the Sec61 complex. Saturation binding of RNC complexes to Sec61 oligomers will indicate whether the SR interferes with the non-specific binding of ribosomes to adjacent vacant Sec61 complexes. The objective of the second specific aim of this proposal is to test the hypothesis that the SRbeta GTPase performs a crucial role in RNC targeting to the translocation channel. To define a role for SR beta, translocation assays will be conducted using SRP54 and SRalpha mutants that bind xanthosine triphosphate in preference to GTP. Functional assays that monitor individual reaction steps during protein translocation will be used to define the events that are dependent upon the GTP binding and hydrolysis activities of Srbeta. The nucleotide binding status of Srbeta will be evaluated in the context of protein translocation reaction. The GTPase cycle of SRbeta will be analyzed in vitro by identifying the protein factors that regulate ribonucleotide exchange and activate GTP hydrolysis. The objective of the third specific aim of this proposal is to test the hypothesis that cooperative binding of GTP to the SRP-SR complex initiates the transfer of the signal sequences from SRP54 to Sec61alpha. Cooperative binding of GTP to the SRP-SR complex will be evaluated using purified SR and a reconstituted SRP containing a SRP54 mutant that binds xanthosine triphosphate in preference to GTP. Transfer of the signal sequence from SRP54 to the Sec61 complex is blocked in N-ethyl maleimide modified microsomes. Dissociation of SRP from RNC complexes will be investigated using NEW-inactivated microsomes to determine whether dissociation of SRP54 from the signal sequence is contingent upon the presence of an active acceptor for the nascent polypeptide.
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