Substrate Proteins Take Shape at an Improved Bacterial Translocon

Abstract

Characterization of Sec-dependent bacterial protein transport has often relied on an in vitro protein translocation system comprised in part of Escherichia coli inverted inner membrane vesicles or, more recently, purified SecYEG translocons reconstituted into liposomes using mostly a single substrate (proOmpA). A paper published in this issue (P. Bariya and L. Randall, J Bacteriol 201:e00493-18, 2019, https://doi.org/10.1128/JB.00493-18) finds that inclusion of SecA protein during SecYEG proteoliposome reconstitution dramatically improves the number of active translocons. This experimentally useful and intriguing result that may arise from SecA membrane integration properties is discussed here. Furthermore, determination of the rate-limiting transport step for nine different substrates implicates the mature region distal to the signal peptide in the observed rate constant differences, indicating that more nuanced transport models that respond to differences in protein sequence and structure are needed.

Staphylococcus carnosus

“…Since no significant differences were detected, the presence of the staphylococcal L13 protein in E. coli strains did not result in a gross defect of protein synthesis. Although an attractive hypothesis by which the ribosomes containing the S. carnosus L13 homologue have a reduced affinity for a component of the E. coli secretion machinery cannot be excluded, it seems very likely however that, in accordance with previous results [35][36][37], some minor effects on protein synthesis, not detected in our experiments, are responsible for the suppression of the E. coli secretion mutants by the S. carnosus rplM gene product.…”

“…Thus, the recombinant strain contained a mixed = population of ribosomes with a L13 protein derived from either E. coli or S. carnosus. It has been shown in several cases that decreasing the rate of protein synthesis can reverse the phenotypes of sec mutants [35][36][37]; the reduction in the amount of secretory proteins probably allows the defective secretion machinery to fulfil] its essential needs [35]. Therefore, the growth rate and uptake of [35S]-methionine into E. coli strains with or without the S. carnosus rplM gene was measured.…”

“…Earlier studies have shown that decreasing the rate of protein synthesis can suppress the growth defects of temperature-sensitive E. coli secretion mutants [35][36][37]. To address this point, we measured the growth rate and [35S]-methionine incorporation of E. coli strains MC4100 and MM52 containing pHC79 or pBO23, respectively.…”

Characterizing the Locus of a Peripheral Membrane Protein–Lipid Bilayer Interaction Underlying Protein Export Activity in E. coli

“…The complexity of the general secretory system has obfuscated central facets, including the fundamental nature of the translocation mechanism. “Power-stroke”, “Brownian ratchet”, and other mechanisms have been proposed, ,,, and recent experiments have shown aspects of the process that vary with precursor species. − What is clear is that proteins are generated at bacterial ribosomes at a rate of ≈20 amino acids per second . Measured rates for SecA-driven translocation of the precursor of outer membrane protein A through SecYEG range from about 5 to 40 amino acids per second. − The mean bound-state lifetime from our dynamic force spectroscopy experiments of SecA2–11 and E.…”

Direct visualization of the E. coli Sec translocase engaging precursor proteins in lipid bilayers

“…Aspects of the translocation reaction have been observed to vary with the precursor species undergoing transport ( 6 , 13 , 14 ). However, precursor-dependent translocation processes have been largely overlooked in the literature ( 15 ).…”