ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinery
Abstract
Transport of proteins across membranes is a fundamental process, achieved in every cell by the 'Sec' translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide [Allen et al. eLife 2016]. Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogen-deuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.
Data availability
All data generated during this study are included in the Figures of the mansucript. EPR data is available at https://doi.org/10.17630/0fedaeec-7e27-4876-a6d1-cda2d3a6799c.
-
EPR data from ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinerydoi:10.17630/0fedaeec-7e27-4876-a6d1-cda2d3a6799c.
Article and author information
Author details
Funding
Biotechnology and Biological Sciences Research Council (BB/M003604/1)
- Robin A Corey
- William John Allen
- Ian Collinson
Wellcome (104632)
- William John Allen
- Ian Collinson
Royal Society (University Research Fellowship)
- Janet E Lovett
Wellcome (109854/Z/15/Z)
- Zainab Ahdash
- Argyris Politis
Wellcome (099149/Z/12/Z)
- Anokhi Shah
- Janet E Lovett
European Regional Development Fund (CZ.02.1.01/0.0/0.0/15_003/0000441)
- Tomas Fessl
Engineering and Physical Sciences Research Council (ep/m508214/1)
- Anokhi Shah
Biotechnology and Biological Sciences Research Council (BB/I008675/1)
- Robin A Corey
- William John Allen
- Ian Collinson
Biotechnology and Biological Sciences Research Council (BB/N015126/1)
- Robin A Corey
- William John Allen
- Ian Collinson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Corey et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,918
- views
-
- 471
- downloads
-
- 33
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Cell Biology
Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3’ untranslated region (3’-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3’-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3’-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling, but not with translational efficiency. The results presented here show that eIF3 engages with 3’-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Mycofactocin is a redox cofactor essential for the alcohol metabolism of mycobacteria. While the biosynthesis of mycofactocin is well established, the gene mftG, which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes are almost exclusively found in genomes containing mycofactocin biosynthetic genes and are present in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the ∆mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.