Protein Translocation Across the ER Requires a Functional GTP Binding Site in the « 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 a subunit of the SRP receptor (SRa), has indicated that these proteins contain predicted GTP binding sites . Several point mutations confined to the GTP binding consensus elements of SRa were constructed by site specific mutagenesis to define a role for the GTP binding site in SRa during protein translocation . The SRa mutants were analyzed using an in vitro system wherein SRa-deficient microsomal membranes were repopulated with SRa by in vitro translation of wild-type or mutant mRNA transcripts . SRP RBOSOMES synthesizing proteins with signal sequences that specify translocation across the RER are selectively delivered to the membrane by the combined action of the signal recognition particle (SRP)' and the SRP receptor. SRP, a ribonucleoprotein particle composed of the SRP RNA and six polypeptide subunits (Walter and Blobel, 1982), binds to the signal sequence shortly after it emerges from the large ribosomal subunit (Walter and Blobel, 1981 ; Walter et al ., 1981) . Nascent polypeptides containing photoactivatable amino acid analogues can be cross-linked to the 54-kD subunit ofthe SRP (SRP54) (Krieg et al ., 1986 ; Kurzchalia et al ., 1986) . The signal sequence binding site was shown to reside within a carboxyl-terminal methionine-rich domain of SRP54 (Zopf et al ., 1990 ; High and Dobberstein, 1991) as initially postulated by Bernstein et al . (1989) . The SRP-ribosome-nascent polypeptide complex is targeted to the membrane through an interaction with the SRP receptor, or docking protein (Walter and Blobel, 1981 ; Gilmore et al ., 1982b ; Meyer et al ., 1982) . The SRP receptor is a heterodimeric protein with an a subunit of 68 kD (SRa) and a ß subunit of30 kD (8Rß) (Gilmore et al ., 1982b ; Tajima et al ., 1986) . After the SRP receptor mediated dissociation of the 1 . Abbreviations used in this paper: K-RM, SRP-depleted RM ; RM, rough microsomal membranes ; SRP, signal recognition particle ; TS-K-RM, tryp-