PROTEIN TRANSLOCATION ACROSS THE ER REQUIRES A FUNCTIONAL GTP BINDING-SITE IN THE ALPHA-SUBUNIT OF THE SIGNAL RECOGNITION PARTICLE RECEPTOR

The signal recognition particle (SRP)-mediated translocation of proteins across the RER is a GTP dependent process. Analysis of the primary amino acid sequence of one protein subunit of SRP (SRP54), as well as the alpha-subunit of the SRP receptor (SR-alpha), has indicated that these proteins contain predicted GTP binding sites. Several point mutations confined to the GTP binding consensus elements of SR-alpha were constructed by site specific mutagenesis to define a role for the GTP binding site in SR-alpha during protein translocation. The SR-alpha mutants were analyzed using an in vitro system wherein SR-alpha-deficient microsomal membranes were repopulated with SR-alpha by in vitro translation of wild-type or mutant mRNA transcripts. SRP receptors containing SR-alpha point mutants were analyzed for their ability to function in protein translocation and to form guanylyl-5'-imidodiphosphate (Gpp[NH]p) stabilized complexes with the SRP. Mutations in SR-alpha produced SRP receptors that were either impaired or inactive in protein translocation. These SRP receptors were likewise unable to form Gpp(NH)p stabilized complexes with the SRP. One SR-alpha point mutant, Thr 588 to Asn 588, required 50- to 100-fold higher concentrations of GTP relative to the wild-type SR-alpha to function in protein translocation. This mutant has provided information on the reaction step in protein translocation that involves the GTP binding site in the a subunit of the SRP receptor.