Abstract
Membrane proteins in bacteria are cotranslationally inserted into the plasma membrane through the SecYEG translocon. Ribosomes exposing the signal-anchor sequence (SAS) of a membrane protein are targeted to the translocon by the signal recognition particle (SRP) pathway. SRP scans translating ribosomes and forms high-affinity targeting complexes with those exposing a SAS. Recognition of the SAS activates SRP for binding to its receptor, FtsY, which, in turn, is primed for SRP binding by complex formation with SecYEG, resulting in a quaternary targeting complex. Here we examine the effect of SecYEG docking to ribosome-nascent-chain complexes (RNCs) on SRP binding and SAS transfer, using SecYEG embedded in phospholipid-containing nanodiscs and monitoring FRET between fluorescence-labeled constituents of the targeting complex. SecYEG–FtsY binding to RNC–SRP complexes lowers the affinity of SRP to both ribosome and FtsY, indicating a general weakening of the complex due to partial binding competition near the ribosomal peptide exit. The rearrangement of the quaternary targeting complex to the pre-transfer complex requires an at least partially exposed SAS. The presence of SecYEG-bound FtsY and the length of the nascent chain strongly influence nascent-chain transfer from SRP to the translocon and repositioning of SRP in the post-transfer complex.
Highlights
Extracytosolic proteins constitute about one third of the bacterial proteome and there are various pathways to target these proteins to the membrane in order to insert them into the lipid bilayer or translocate them through the membrane
As the signal recognition particle (SRP) receptor FtsY does not bind to the detergent-solubilized translocon[24], the SecYEG translocon was inserted into nanodiscs containing a mixture of phospholipids characteristic for the plasma membrane of E. coli[16,24,26]; the preparation yields nanodiscs containing a single copy of SecYEG, as verified by quantitative mass spectrometry (Material and Methods), that was functionally competent[16,21]
We examined how the initial quaternary targeting complex rearranges to a pre-transfer complex which consists of an ribosome-nascent-chain complexes (RNCs) with bound SRP and the SecYEG translocon with bound FtsY and upon transfer of the signal-anchor sequence (SAS) to a post-transfer complex (Fig. 6)
Summary
Extracytosolic proteins constitute about one third of the bacterial proteome and there are various pathways to target these proteins to the membrane in order to insert them into the lipid bilayer or translocate them through the membrane. On binding to the translocon in the membrane, FtsY is activated by an engagement of the A domain with SecYEG and membrane phospholipids[16,17] This frees the NG domain of FtsY for the interaction with the homologous NG domain of SRP protein Ffh[18]. These activation events lead to an increased affinity of SRP binding to FtsY and take place at the ribosome or at SecYEG1,2,16,19,20. The increased affinity between SRP and FtsY and between RNC and SecYEG favors the assembly of the quaternary targeting/transfer complex at the translocon[21,22]. We determined changes of the affinity of the SRP–FtsY complex in the course of targeting and transfer of the RNC and the handover of the SAS from SRP to SecYEG applying FRET and proteinase protection assays
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