A signal-anchor sequence stimulates signal recognition particle binding to ribosomes from inside the exit tunnel

Abstract

Sorting of eukaryotic membrane and secretory proteins depends on recognition of ribosome-bound nascent chain signal sequences by the signal recognition particle (SRP). The current model suggests that the SRP cycle is initiated when a signal sequence emerges from the ribosomal tunnel and binds to SRP. Then elongation is slowed until the SRP-bound ribosome-nascent chain complex (RNC) is targeted to the SRP receptor in the endoplasmic reticulum (ER) membrane. The RNC is then transferred to the translocon, SRP is released, and translation resumes. Because RNCs do not target to the translocon efficiently if nascent chains become too long, the window for SRP to identify its substrates is short. We now show that a transmembrane signal-anchor sequence (SA) significantly enhances binding of SRP to RNCs even before the SA emerges from the ribosomal tunnel. In this mode, SRP does not contact the SA directly but is in close proximity to the portion of the nascent polypeptide that has already left the ribosomal tunnel. Early recruitment of SRP provides a mechanism to expand the window for substrate identification. We suggest that the dynamics of the SRP-ribosome interaction is affected not only by the direct binding of SRP to an exposed signal sequence but also by properties of the translating ribosome that are triggered from within the tunnel.chaperones ͉ site-specific cross-linking ͉ yeast ͉ nascent polypeptide-associated complex ͉ Ssb1/2

Elongation Factor 1A Is the Target of Growth Inhibition in Yeast Caused by Legionella pneumophila Glucosyltransferase Lgt1

“…Transcription was performed using SP6 polymerase (22) and pSP-lucϩ (Promega) as a template to generate firefly luciferase mRNA. Translation reactions were performed as described (22)(23)(24). To test for the ability of Lgt1 to inhibit translation in yeast system, purified His-tagged Lgt1 at 9, 90, or 280 nM final concentrations and 10 M UDPglucose as a Lgt1 co-substrate were added to the translational mixes lacking luciferase mRNA.…”

Cytosolic targeting factor AKR2A captures chloroplast outer membrane-localized client proteins at the ribosome during translation

“…This is an elegant mechanism for client protein sorting by factors involved in organellar protein targeting. This process is reminiscent of SRP action in ER targeting, in which SRP-binding affinity for ribosomes is greater when ribosomes are engaged in translation of SRP clients 50 .…”

“…The TMD of COM proteins has lower hydrophobicity than that of ER proteins 52 . Recent studies have suggested that the presence of a signal-anchor sequence within the exit tunnel of the ribosome promotes binding of SRP to the ribosome 50 . The dynamics of the SRP-ribosome interaction is affected by the biophysical properties of the nascent polypeptide located within the exit tunnel of the translating ribosome.…”

“…The primer sequences used in this study are listed in Supplementary Table 5. The primers used were as follows: RPL23aA-F1 and RPL23aA-R1 for His:RPL23aA; RPL23aB-F1 and RPL23aB-R1 for His:RPL23aB; RPL32A-F and RPL32A-R for His:RPL32A; RPL23aA-F2 and RPL23aA-R2 for GST:RPL23aA; RPL23aB-F2 and RPL23aB-R2 for GST:RPL23aB; OEP7-F1 and OEP7(9)-R for GST:OEP7(9); OEP7-F1 and OEP7(27)-R for GST:OEP7(27); OEP7-F1 and OEP7(36)-R1 for GST:OEP7(36); and OEP7-F1 and OEP7(50)-R1 for GST:OEP7 (50). PCR products were ligated to pRSET-A for His tagging or pGEX5x1 for GST tagging.…”

“…The OEP7 targeting signal contains two components, TMD (aa positions 10 À 27) and CPR (aa positions 28 À 36). We generated four serial deletion mutants designated OEP7(9), OEP7 (27), OEP7(36) and OEP7 (50). OEP7 (9) did not contain the targeting signal, OEP7 (27) contained only TMD but lacked CPR, and OEP7(36) and OEP7(50) contained both TMD and CPR.…”