Rate-limiting transport of positively charged arginine residues through the Sec-machinery is integral to the mechanism of protein secretion

  1. William J Allen
  2. Robin A Corey
  3. Daniel W Watkins
  4. A Sofia F Oliveira
  5. Kiel Hards
  6. Gregory M Cook
  7. Ian Collinson

  • Curated by eLife

    eLife logo

    Evaluation Summary:

    Here, using a novel bioluminescence-based assay, authors dissect the sequence features of client proteins that influence SecA/SecYEG-mediated protein translocation across the bacterial inner membranes. Combined with rigorous kinetic modeling, this study pushes the description of this important cellular pathway towards a highly detailed level, which will potentially advance our understanding of ATP-driven protein secretion mechanisms in bacteria. The main conclusions are well supported, and the paper will be interesting to both those in the field of protein transport and to a broader audience.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Transport of proteins across and into membranes is a fundamental biological process with the vast majority being conducted by the ubiquitous Sec machinery. In bacteria, this is usually achieved when the SecY-complex engages the cytosolic ATPase SecA (secretion) or translating ribosomes (insertion). Great strides have been made towards understanding the mechanism of protein translocation. Yet, important questions remain – notably, the nature of the individual steps that constitute transport, and how the proton-motive force (PMF) across the plasma membrane contributes. Here, we apply a recently developed high-resolution protein transport assay to explore these questions. We find that pre-protein transport is limited primarily by the diffusion of arginine residues across the membrane, particularly in the context of bulky hydrophobic sequences. This specific effect of arginine, caused by its positive charge, is mitigated for lysine which can be deprotonated and transported across the membrane in its neutral form. These observations have interesting implications for the mechanism of protein secretion, suggesting a simple mechanism through which the PMF can aid transport by enabling a 'proton ratchet', wherein re-protonation of exiting lysine residues prevents channel re-entry, biasing transport in the outward direction.

Article activity feed

  1. Version published to 10.7554/elife.77586 on eLife
  2. eLife

    Evaluation Summary:

    Here, using a novel bioluminescence-based assay, authors dissect the sequence features of client proteins that influence SecA/SecYEG-mediated protein translocation across the bacterial inner membranes. Combined with rigorous kinetic modeling, this study pushes the description of this important cellular pathway towards a highly detailed level, which will potentially advance our understanding of ATP-driven protein secretion mechanisms in bacteria. The main conclusions are well supported, and the paper will be interesting to both those in the field of protein transport and to a broader audience.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

  3. eLife

    Reviewer 1 (Public Review):

    In this work Allen and colleagues set out to dissect the mechanisms of protein transport through the Sec machinery. The authors implement the NanoLuc assays, MD simulations, and bioinformatics to sketch out a conceptually interesting hypothesis on protein secretion. The findings suggest that pre-proteins move through the Sec machinery by diffusion and that transport of arginine residues accounts for most of the transport time. The conclusions are supported by the data. Overall, this is an interesting paper on a fundamental topic that will be of interest to a wide audience.

  4. eLife

    Reviewer 2 (Public Review):

    In this study, authors tested the effects of various sequence features of a client preprotein (e.g., the charges, hydrophobicity, helix propensity, and residue sizes) as well as of the proton-motive force on the SecA/SecYEG-mediated protein transport. The experimental designs are elegant, and the results are interesting. Especially, the role of Arg residues in decelerating protein transport is notable. Authors corroborate on explaining the negligible impact of Lys on the rate. The explanation based on MD simulation is also reasonable. Using the sophiscated kinetic model and analysis, authors propose a detailed protein transport mechanism supporting the ratcheting model, which is impressive.

  5. eLife

    Reviewer 3 (Public Review):

    The Sec system transports proteins across bacterial membranes and has relevance to myriad biological processes. A common problem in biochemistry is the lack of a good assay for the particular activity of interest and this affected the study of Sec. However, recently, the Collinson lab developed a high-resolution assay for transport, using a split NanoLuc luciferase, where '11S' (the majority of the NanoLuc enzyme) has a small ß-strand removed ('pep86'). When this pep86 is complemented back, the enzyme is active and a luminescence signal is generated allowing transport to be monitored. Previously, this system was used to perform a high-resolution kinetic analysis, a major first for the field, which set the scene for this work.

    Here, the authors have built upon the split NanoLuc system to explore the details of the interaction between the charge of the protein substrate and kinetics of transfer. To do this, they created a model substrate using three tandem pSpy domains, the pep86 signal, and the signal sequence. They then modified the sequence of the central domain to mutate different amino acids to alter bulkiness and charge and observe that of all the mutations, introducing positive arginine residues slows transport the most. Here, the data are convincing, with clear trends being observed. The authors then modify their previously published kinetic modelling to determine step size. They find that for nearly all of the variants, their model predicts about the same number of steps apart from the arginine-containing one, where the model breaks down and does not provide a unique solution. The authors postulate that of the two positive amino acids, arginine presents the most difficulty in transport as its pKa is much too high to be deprotonated before transport, whereas lysine may be as the pKa will be lowered in the hydrophobic interior of the translocon. This idea is both intuitive and supported with careful molecular dynamics simulations. The observation that the immutable positive charge on arginines presents intrinsic difficulties to transport is further supported by a bioinformatics analysis by comparing Tat vs Sec mediated transport and by comparing Bacillus halodurans with Bacillus subtilis; as these organisms grow at different pH values their ΔpH should be radically different, which should affect the ease of transporting arginine residues. Finally, inverted vesicle systems are used to measure the transport of the model substrates while a PMF is established through ATP hydrolysis. Here the data are less clear cut and and it appears that PMF has a major effect on transport depending on pre-protein hydrophobicity (rather than depending on charged residues as one might expect). This is an intriguing finding to end the paper on and I'm sure is something that the authors can build upon in the future.

    Overall, the paper is a seminal advance in understanding the details of protein transport through the Sec translocon. The authors are careful to caveat their statements throughout so as to not overstate the strength of their findings and honestly report both clear and 'messy' observations.

  6. Version published to 10.1101/592543v2 on bioRxiv
  7. Version published to 10.1101/592543v1 on bioRxiv