Co‐translational insertion and topogenesis of bacterial membrane proteins monitored in real time

Abstract

Integral membrane proteins insert into the bacterial inner membrane co‐translationally via the translocon. Transmembrane (TM) segments of nascent proteins adopt their native topological arrangement with the N‐terminus of the first TM (TM1) oriented to the outside (type I) or the inside (type II) of the cell. Here, we study TM1 topogenesis during ongoing translation in a bacterial in vitro system, applying real‐time FRET and protease protection assays. We find that TM1 of the type I protein LepB reaches the translocon immediately upon emerging from the ribosome. In contrast, the type II protein EmrD requires a longer nascent chain before TM1 reaches the translocon and adopts its topology by looping inside the ribosomal peptide exit tunnel. Looping presumably is mediated by interactions between positive charges at the N‐terminus of TM1 and negative charges in the tunnel wall. Early TM1 inversion is abrogated by charge reversal at the N‐terminus. Kinetic analysis also shows that co‐translational membrane insertion of TM1 is intrinsically rapid and rate‐limited by translation. Thus, the ribosome has an important role in membrane protein topogenesis.

Residue-by-residue analysis of cotranslational membrane protein integration in vivo

“…We have previously shown that a model TMH composed of Ala and Leu residues generates a peak in an FP recorded with the SecM( Ec ) AP that reaches half-maximal amplitude ( N start ) when the N-terminal end of the TMH is ~45 residues away from the polypeptide transferase center (PTC) ( Ismail et al, 2012 ), and a recent real-time FRET study of cotranslational membrane integration found that the N-terminal end of the first TMH in a protein reaches the vicinity of the SecYEG translocon when it is 40–50 residues away from the PTC ( Mercier et al, 2020 ). For EmrE(C out ) TMH1, this would correspond to constructs with N ≈ 50.…”

Translational Control by Ribosome Pausing in Bacteria: How a Non-uniform Pace of Translation Affects Protein Production and Folding

“…Membrane proteins were shown to have multiple pauses during translation in vivo ( Chadani et al, 2016 ). They have long clusters of rare codons that may play a role in defining the time window for correct targeting and membrane insertion of these proteins ( Zalucki and Jennings, 2007 ; Chartier et al, 2012 ; Mercier et al, 2020 ). The topology of transmembrane helices generally follows the “positive-inside” rule, according to which the distribution of positively charged residues at the N-terminus of the bacterial inner membrane proteins determines the orientation of the first transmembrane helix with its N-terminus in the cytoplasm or the periplasm ( von Heijne, 1989 ).…”

“…However, computer simulations suggested that variations in the translation rate may alter transmembrane helix topology ( Zhang and Miller, 2012 ; Niesen et al, 2017 ), indicating that the “positive-inside” rule is not the only driver of correct membrane insertion. In fact, a recent biochemical study which monitored cotranslational insertion of transmembrane segments of E. coli EmrD protein, showed that a prolonged ribosome stalling at a critical position can alter the topology of transmembrane segments ( Mercier et al, 2020 ), underscoring the important link between the rate of translation and protein biogenesis and folding.…”

Residue-by-residue analysis of cotranslational membrane protein integrationin vivo

“…4e. Upstream positively charged residues thus delay the membrane integration of the Nout-oriented TMH2, possibly because of the energetic cost of translocating them against the membrane potential (3), or because they are temporarily retained in the negatively charged exit tunnel (15). The low Nstart values for peaks X and XI are likely caused by the short upstream hydrophobic segments LCGL and LAAALEL (Fig.…”

“…Point mutations in an upstream TMH can affect the pulling force generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the membrane-integration process. Complementing in vitro unfolding/folding studies (27,28), real-time FRET analyses (15), chemical crosslinking (29), structure determination (30), and computational modeling (31), high-resolution in vivo FPA can help identify the molecular interactions underlying cotranslational membrane protein biogenesis with single-residue precision. For constructs with N≥298, the C-terminal tail is 75 residues long.…”