GTP hydrolysis by complexes of the signal recognition particle and the signal recognition particle receptor.

Abstract

Abstract. Translocation of proteins across the endoplasmic reticulum membrane is a GTP-dependent process. The signal recognition particle (SRP) and the SRP receptor both contain subunits with GTP binding domains. One GTP-dependent reaction during protein translocation is the SRP receptor-mediated dissociation of SRP from the signal sequence of a nascent polypeptide. Here, we have assayed the SRP and the SRP receptor for GTP binding and hydrolysis activities. GTP hydrolysis by SRP was not detected, so the maximal GTP hydrolysis rate for SRP was estimated to be <0.002 mol GTP hydrolyzed x mol of SRP -1 x min-'. The intrinsic GTP hydrolysis activity of the SRP receptor ranged between 0.02 and 0.04 mol GTP hydrolyzed × mol of SRP receptor -l x min -1. A 40-fold enhancement of GTP hydrolysis activity relative to that observed for the SRP receptor alone was obtained when complexes were formed between SRP and the SRP receptor. GTP hydrolysis activity was inhibited by GDP, but not by ATE Extended incubation of the SRP or the SRP receptor with GTP resulted in substoichiometric quantities of protein-bound ribonucleotide. SRP-SRP receptor complexes engaged in GTP hydrolysis were found to contain a minimum of one bound guanine dbonucleotide per SRP-SRP receptor complex. We conclude that the GTP hydrolysis activity described here is indicative of one of the GTPase cycles that occur during protein translocation across the endoplasmic reticulum.

Membrane Binding of Ribosomes Occurs at SecYE-based Sites in the Archaea Haloferax volcanii

“…At the onset of co-translational protein translocation in Eukarya, ribosomes in the process of synthesizing signal peptide-bearing nascent polypeptides are recognized by SRP and delivered to the ER membrane via the affinities of SRP for its receptor 12,13 and of the ribosome for the Sec61 complex. 14,15 Similarly, bacterial ribosomes have been shown to specifically bind to SecYEG complexes, 20,21 although the physiological significance of this finding remains the subject of investigation.…”

“…10 SRP binding subsequently slows or prevents the continuation of further protein synthesis. 11 The ternary complex of ribosome -nascent polypeptide chain -SRP then interacts with the ER membrane via the affinities of SRP for the membrane-associated SRP receptor, 12,13 but also via the interaction between the ribosome and Sec61abg, the core of the translocation complex (the "translocon"). 14,15 Once delivered to the translocon, the arrest of protein synthesis mediated by SRP is removed and synthesis of the nascent chain into the translocon, and hence, across the ER membrane, proceeds.…”

The Srp54 GTPase is essential for protein export in the fission yeast Schizosaccharomyces pombe.

“…Using an in vitro membrane repopulation assay, it has been shown that site-specific mutations in the GTPase consensus motifs of mammalian SRa disrupt its function (71). Although photoaffinity labeling in vitro demonstrates that Srp54 and both subunits of the SRP receptor are GTP-binding proteins (20), GTP hydrolysis by mammalian Srp54 could be detected only in the presence of the SRP receptor and SRP RNA (21,53,54,66). In contrast, the M. mycoides Srp54 protein displays readily detectable GTPase activity in the absence of other factors (81).…”

Anionic Phospholipids and the Albino3 Translocase Activate Signal Recognition Particle-Receptor Interaction during Light-harvesting Chlorophyll a/b-binding Protein Targeting

“…At the membrane, the SRP⅐SR complex unloads its cargo to the SecYEG or Sec61p translocase, which mediates the integration or translocation of the nascent protein across the membrane (9,10). In addition, GTP hydrolysis is activated in the SRP⅐SR complex to drive their disassembly, allowing SRP and SR to enter the next round of targeting (11)(12)(13).…”