the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 10 Mar 2026
Bioengineering the Ice Worm Complex V ATP6 subunit to Increase Energy Production
Abstract. Glacier ice worms, Mesenchytraeus solifugus, are among a few animals that complete their life cycle in hydrated glacier ice. These worms are distinguished from congener species by relatively high intracellular ATP levels, likely associated with a lateral gene transfer event adding 18 amino acids (aa) to the carboxy terminal of the Complex V FO ATP6 subunit, a major regulator of ATP synthesis. By examining the kinetic profiles of respective 13/18 aa natural variants fused with E. coli AtpB (prokaryotic counterpart of ATP6) in the context of ice worm evolution, we conclude that the 18 aa variant predates the 13 aa variant, but the latter likely outcompetes the former in natural populations. Utilizing the 13 aa variant as a template, a panel of genetically engineered extension mutants were constructed and tested by Michaelis–Menten kinetics as a function of increasing [ADP]. Our data show that Vmax can be increased significantly over natural 13/18 aa variants, and suggest a functional mechanism by which a dynamic tether transfers protons from the FO exit pore to the mitochondrial lumen via the protonation/deprotonation of distal histidine residue(s).
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Status: open (until 15 Aug 2026)
- RC1: 'Comment on egusphere-2026-1073', Anonymous Referee #1, 12 Jun 2026 reply
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RC2: 'Comment on egusphere-2026-1073', Anonymous Referee #2, 16 Jun 2026
reply
In their simulations, authors intended to study the mitochondria-encoded ATP6 subunit of ATP-synthase of glacier ice worms containing 13 aa and 18 aa extensions. Mimicking this eukaryotic enzyme by genetically engineered 13 aa and 18 aa variants added to E. coli AtpB sequence and assaying Michaelis Menten kinetics dependent on ADP, they interpreted the observed Vmax increase as being due to protonation deprotonation of distal histidine residues. This function was supported in the current manuscript by demonstration that the increased Vmax depends precisely on spatial positions of histidines rather than on their number. Nevertheless, the precise nature of mechanism for enhanced ATP production was not definitively characterized.
Since authors reached an innovation threshold by demonstrating that various mutants have different Vmax in the mimicking reconstituted system, the manuscript can be recommended for publication.
Citation: https://doi.org/10.5194/egusphere-2026-1073-RC2 -
RC3: 'Comment on egusphere-2026-1073', Ryan Steed, 14 Jul 2026
reply
Mutlu, Dunkley, and Shain expand on previous work showing that the histidine-rich C-terminal extension on F1Fo ATP synthase subunit a (aka ATP6) is associated with increased ATP synthesis activity. In the current study, the authors characterize additional variants of the subunit a extension, both naturally occurring and engineered, attached to E. coli ATP synthase. Inverted membrane vesicles prepared from these mutant E. coli appear to show ATP synthesis activity with an increased vmax. If these results are validated, then the additional constructs contribute to a molecular mechanistic explanation of the increased activity, which the authors posit is increasing the rate of deprotonation in the N-side proton exit channel. The interesting results reported here have the potential to impact our understanding of a divergent mechanism of proton transport in ATP synthase. To maximize the impact, the authors should address two missing controls and clarify their data analysis.
Major Comments
- Preparations of inverted membrane vesicles from bacteria often contain background levels of ATP and ATP synthesis activity independent of F1Fo ATP synthase, e.g. adenylate kinase, which must be subtracted to accurately determine the ATP synthesis activity of the engineered enzymes. The authors should measure ATP production in each of their vesicle preparations in the presence of a protonophore, like carbonyl cyanide m-chlorophenylhydrazone (CCCP), to determine the gradient-independent background. Subtraction of this background would be essential prior to normalization of activity to expression.
- The Western blots suggest that atpB mutants are expressing much lower amounts of subunit a. The authors postulate that mutants, being hyperactive, can sustain cells with lower expression, but the data do not necessarily support this conclusion. The mutant subunit a may be unstable or less able to assemble the Fo complex. To support their conclusion, the authors should determine whether their mutant E. coli strains can grow aerobically in non-fermentable minimal medium at the same level as wildtype.
- The normalization of ATP synthesis activity to subunit a expression level is understandable (and key to the authors’ conclusion). The authors need to ensure that their analysis is completely transparent:
- The Western blots (Fig. A1) show significant variability across biological replicates. Please clarify in the text how band intensities are used for normalization. Are they compared to the intensities of LamB? In section 2.6, line 164-166, it suggests that the intensities of WT bands are somehow used for normalization.
- When expression level is very low (Mutants 3 and 6), amplification of error upon normalization would be significant. Additionally, background ATP synthesis activity (see Comment 1) would be significantly amplified. It may be useful to include the unnormalized activity values and error in a supplementary table.
- If the ATP synthesis activities are normalized to subunit a level using relative density on a Western blot, then this value is no longer an absolute specific activity, making the y-axis label (nmol ATP/s/mg mem prot) in Figures 1 and 3 misleading. It would be more accurately reported as specific activity relative to whatever intensity (WT? LamB?) was used for normalization.
Citation: https://doi.org/10.5194/egusphere-2026-1073-RC3
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Summary:
This manuscript characterizes the function of ATP synthase containing the C-terminal 13/18 aa extension variants found in Mesenchytraeus solifugus ATP6. This work shows that fusion protein of E. coli AtpB containing the 13 aa extension from M. solifugus confers increased ATP synthesis rate in a similar manner as the 18 aa extension fusion protein shown in a previous study. The authors also explored the ATP synthesis activities of engineered mutants derived from the 13 aa variant sequence, in which alternating histidine residues were added or substituted in the sequence. The results showed that some of the engineered mutants outperform the wild-type 13 aa variant.
The authors conclude that the presence of histidine residues and the flexibility of the extension region contribute to an increase in proton transfer by the FO motor, which results in the increased ATP synthesis rate. This conclusion is consistent with and provides significant insight into the unique characteristic of M. solifugus, in which the ATP level is higher compared to similar species. The increased ATP synthesis rate of the engineered mutants contributes to the overall understanding of the enzyme mechanism, and also has biomedical implications.
Overall, the strengths of this work are the clear demonstration that ATP6 C-terminal 13 aa extension causes elevated ATP synthesis rate, consistency of the results with the observed high ATP production by M. solifugus, and the mutagenesis work that characterizes how the histidine-rich extension region enhances the ATP synthesis rate. The major weakness of this study is the quality of the Western blot for Mutants 3 and 6 and the reliance on that result to conclude that those two mutants have the highest ATP synthesis rates, which must be addressed before publication.
Major comments
1. Mutants 3 and 6 are shown to have the highest normalized ATP synthesis rates. However, these are the two mutants that showed very faint Western blot bands that were almost faded to the background (supplementary figure A1). Therefore, it is concerning whether the ATP synthesis rates for these mutants were accurate upon normalization. If the ATP synthesis activities of Mutants 3 and 6 are not accurate, the conclusions made by the authors in lines 308 and 328 (regarding flexibility being more critical than the number of histidine residues) must be re-evaluated.
To address this, the authors should provide:
2. Line 491 - The authors suggest that Mutants 3 and 6 had lower expression of ATP synthase because “fewer ATP synthase complexes were necessary to meet cellular energy demands”. It is also possible that expression levels were low because the mutant protein was unstable, not because the cell did not need as many ATP synthase complexes.
To address this, the authors should provide:
3. To truly substantiate the mechanism proposed in the final paragraph, a proton flux assay (such as an ACMA quenching assay) would directly quantify the relationship between the increased ATP synthesis rates and the proton transfer rates through the FO This would significantly add to the mechanistic understanding of the unique characteristics of this ATP synthase species and the function of the FO motor in general. However, it is understandable if this is outside of the scope of this study.
4. Line 286 – The authors suggest that the 13 aa variant became dominant over the 18 aa in certain regions through natural selection for smaller mitochondrial genome size. However, it is not clear if the 15 bp difference in mitochondrial genome size could impose a significant selective pressure. The authors should acknowledge genetic drift as an equally plausible mechanism for fixation in this section.
Minor Comments: