Signal sequence recognition and targeting of ribosomes to the endoplasmic reticulum by the signal recognition particle do not require GTP.
The identification of GTP-binding sites in the 54-kDa subunit of the signal recognition particle (SRP) and in both the alpha and beta subunits of the SRP receptor has complicated the task of defining the step in the protein translocation reaction that is controlled by the GTP-binding site in the SRP. Ribonucleotide binding assays show that the purified SRP can bind GDP or GTP. However, crosslinking experiments show that SRP54 can recognize the signal sequence of a nascent polypeptide in the absence of GTP. Targeting of SRP-ribosome-nascent polypeptide complexes, formed in the absence of GTP, to microsomal membranes likewise proceeds normally. To separate the GTPase cycles of SRP54 and the alpha subunit of the SRP receptor (SR alpha), we employed an SR alpha mutant that displays a markedly reduced affinity for GTP. We observed that the dissociation of SRP54 from the signal sequence and the insertion of the nascent polypeptide into the translocation site could only occur when GTP binding to SR alpha was permitted. These data suggest that the GTP binding and hydrolysis cycles of both SRP54 and SR alpha are initiated upon formation of the SRP-SRP receptor complex.
Rapiejko
P J
PJ
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01655.
Gilmore
R
R
D000818:Animals; D001665:Binding Sites; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D066298:In Vitro Techniques; D008861:Microsomes / Q000378:metabolism; D009154:Mutation; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D018271:Signal Recognition Particle / Q000378:metabolism*
D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D018271:Signal Recognition Particle; C036318:signal peptide receptor; D006160:Guanosine Triphosphate