Sequence adaptations satisfy the constraints of mitochondrial membrane protein evolution

Reviewer #1 (Public review):

In this manuscript, Yadav and coworkers investigate hydrophobicity governing membrane insertion of mitochondria-encoded polypeptides. The basis of their considerations is the question of why mitochondria maintain a co-translational insertion mechanism and what rules allow allotopic expression. The issue if mitochondrial-encoded proteins can be expressed in the cytoplasm and imported into mitochondria is a long-standing topic in the field with broad implications for the treatment of mitochondrial disorders. In the manuscript, Yadav et al. propose that aliphatic-to-threonine substitution (ATS) are a recurrent and convergent adaptation in the transmembrane helices (TMH) of the inner mitochondrial membrane (MIM) proteins, solving the conflict between hydrophobicity required for membrane insertion and the need for soluble cytosolic transit. The study combines structural comparative genomics, MD simulations, and cellular experiments.

This is a well-written and highly interesting study that provides a solid basis for considerations regarding mitochondrial protein insertion. I am sure that the study will represent an important asset for future studies on allotopic protein expression and potentially the development of genetic tools. The study provides solid evidence, from evolutionary correlation to experimental manipulation and molecular-level explanation. The study provides abundant and well-thought-out controls.

Specific points

(1) There are multiple comparisons throughout the manuscript, and not all of them appear to have been explicitly corrected. Numerous Wilcoxon tests, t-tests, and ANOVAs are reported (Figures 2, 3, 4), without a systematic correction for multiple testing or clear mention of FDR/Bonferroni corrections, except for the directional analysis of substitutions, for which the Benjamini-Hochberg procedure was applied. Given the large number of pairwise comparisons in Figures 3 A-C; and Figure 3 Supplement 1, Supplement 2, Supplement 4, and Supplement 5, with dozens of p-values per panel, it would be relevant to clarify whether a multiple-testing correction was also applied in these analyses. If not, the authors should explain the rationale for not applying one.

(2) The authors used experimental structures as one criterion but, in other cases, AlphaFold2 to compare evolutionarily distant pairs. Using non-experimental structures as a base for structural alignment among very divergent proteins is understandable but also prone to errors, especially for membrane proteins. It might wrongly identify which helices are considered "related". To address this, the authors need to clearly indicate which data are based on experimental or computational data - this is not always the case.

(3) The Kyte-Doolittle hydrophobicity scale is standard but relatively raw in comparison with scales based on experimental insertion energy (like Hessa et al., cited in the text or Bothelo et al.). It might be useful to explain why the authors preferred K-D over an experimental membrane insertion scale for the analysis. Particularly, since the Hessa et al. scale was later used in Figure 8G for the thermodynamic analysis.

(4) The phylogenetic tree of ATP6 (Figure 5A) presents low bootstrap values in some internal nodes (43, 40, 55) on which the interpretation of "independent events" of genetic transfer relies. This must be discussed, since a bootstrap of 40-50% provides not too much confidence in the deep topology of the tree, even though the point that the nuclear sequences don't form a monophyletic group with the mitochondrion seems robust (supported by distinct MTS in each clade).

(5) Definition of "core" vs "peripheric" in TMH (11 central positions) seems arbitrary; it would be worth clarifying why that cut was chosen and whether the results are robust when they vary.

(6) There is a notable gap in the literature when describing some of the findings in the Results and Discussion section:

Biophysics of threonine/serine in TMH:
Senes et al., 2004, Dawson et al., 2002. The result described in Figure 8 (Threonine rotamers) is essentially a biophysical refinement of a body of work already established by the Engelman and DeGrado labs about how Ser-Thr, in comparison with other polar residues, promotes specifically the packing and stability of TMH through intra-inter helical hydrogen bonds, without compromising the membrane insertion. Chamberlain & Bowie, 2002 was included (C-H bonds), and Hessa et al. 2005 (energy of insertion), but no reference was included about why Ser/Thr are polar amino acids "tolerated" at the hydrophobic core of TMH, which is the state the authors describe.

In the argument about why TMH hydrophobicity matters for targeting (not only for membrane insertion), there is much more direct and recent mechanistic work that should be included: Botelho et al., 2011 established experimentally the hydrophobicity threshold for the "stop-transfer" pathway of TIM23 with quantitative data in an analogous way as reported in this work. Lee et al., 2020 and Yang et al., 2025 described structurally how hydrophobicity of the substrate modulates the Mgr2-Tim17 interaction to allow precursors to be embedded within the membrane.

It may be helpful to consider previous published work as an important precedent for the discussion of TMH hydrophobicity and TIM23 in allotopic expression. The final observations of incomplete mitochondrial localization despite reduced TMH hydrophobicity could also be discussed in light of previous published studies. These studies suggest that hydrophobicity is an important factor, but not the only determinant of efficient mitochondrial targeting. The role of TMH hydrophobicity in mitochondrial import had already been explored in this context. This would help clarify how the current findings build upon the existing literature.

Reviewer #2 (Public review):

Summary:

The paper by Yadav et al. provides compelling theoretical and experimental evidence for an aliphatic-to-threonine substitution (ATS) in the transmembrane helices (TMs) of membrane proteins residing in the inner mitochondrial membrane (IMM) across several species. Furthermore, using ATS, they designed variants of the human Atp6 subunit and of Atp6 subunits from other species that seem to be internalized into mitochondria.

Strengths:

In general, the results support the conclusions. The findings are particularly relevant to the field of allotopic expression and could have significant implications for the future development of gene therapies for mitochondrial disorders.

Weaknesses:

The manuscript could be substantially strengthened by the addition of further experimental evidence and by some minor revisions and adjustments to the interpretation and presentation of the results.

Major Points

(1) The protein product of atp6 is an essential component of the proton channel of ATP synthase, and more than a dozen pathogenic mutations in this gene have been associated with mitochondrial diseases. Accordingly, several studies have reported attempts to achieve allotopic expression of Atp6 in human cells. I would dedicate a pair of paragraphs in the Discussion to acknowledging the most relevant of these previous efforts. Here, in the work of Yadav et al., several Atp6 constructs were detected within mitochondria by fluorescence microscopy. However, no direct evidence was provided that the allotopically expressed Atp6 protein was incorporated into complex V. Localization within mitochondria, by itself, does not demonstrate successful allotopic expression in the functional sense, as the protein could remain unassembled, inserted in an incorrect topology, or in a process of degradation. In my view, this is the main weakness of the present study. A convincing demonstration of allotopic ATP6 expression should ideally be supported by biochemical evidence showing that the imported subunit is stably incorporated into ATP synthase and, most importantly, contributes to its function. This would require demonstrating at least partial restoration of the respiratory or OXPHOS defect.

I therefore encourage the authors to test their different constructs in cybrid cells carrying a pathogenic mutation in the mitochondrial atp6 gene and determine whether they can restore OXPHOS function or, at a minimum, respiratory capacity. Such an experiment would provide substantially stronger evidence that the imported Atp6 protein is not merely localized to mitochondria but is functionally incorporated into ATP synthase. Alternatively, Blue Native PAGE of mitochondria isolated after allotopic expression of the different constructs, followed by second-dimensional SDS-PAGE, could provide direct evidence that a tagged Atp6 subunit has been incorporated into the ATP synthase complex. Furthermore, this experiment could provide a valuable functional comparison of the three ATS constructs (T1, T2, and T3) and identify which one is most effective in restoring function. Importantly, the T3 construct, despite carrying the highest level of ATS, may not necessarily be the most effective in increasing the levels of assembled ATP synthase or in restoring ATP synthase activity.

(2) I do not agree with the interpretation of the results presented in Figure 7 - Figure Supplement 3, "Targeting tracks with hydrophobicity, not expression." I am concerned that the experimental design may be biased. The two most highly hydrophobic proteins (WT and T1), which are expected to have greater difficulty being imported into mitochondria, were selected for low-level expression. In contrast, the two constructs with extensive ATS (T2 and T3) were expressed at high levels. The hydrophobicity effect and the expression levels are confounded in this experiment, making it difficult to determine whether the observed targeting differences are attributable to one of these parameters. To disentangle these effects, this experiment should be repeated using both low and high expression levels for all four constructs, resulting in eight experimental conditions (four constructs x two expression levels).

Reviewer #3 (Public review):

Yadav et al. revisit the old conundrum of why certain genes encoding inner mitochondrial membrane (IMM) proteins are retained in the mitochondrial genome (mtIMMs; 13 genes in humans) while genes encoding other IMM proteins (nuIMMs) have been relocated to the nuclear genome during evolution. It was noted already in 1986 that nuIMMs have transmembrane helices (TMHs) of lower hydrophobicity than mtIMMs (FEBS Lett 198:1). A number of subsequent studies have shown that some mtIMMs can be converted to nuIMMs by appending a mitochondrial targeting sequence (MTS) and, in most cases, reducing the hydrophobicity of one or more of the TMHs (reviewed in Genes & Dis 7:578). Conversely, increasing the hydrophobicity of a TMH in a nuIMM protein can prevent its import into mitochondria (e.g., PNAS 99:10510). Whether the reduction in TMH hydrophobicity in nuIMMs is mainly driven by the need to bypass cotranslational targeting of the protein to the ER and trafficking along the secretory pathway, or to prevent the protein from being prematurely stuck in the IMM during mitochondrial import, is still, to some extent and open question, and experimental support for both possibilities exists (e.g., FEBS Lett 591:65).

Yadav et al. present an extensive bioinformatics study which nicely confirms the reduced hydrophobicity of nuIMM TMHs compared to mtIMM TMHs (and also compared to TMHs in proteins that traffic along the secretory pathway). The study is based on a lot more sequence data and many more kinds of comparisons than previous publications, and leaves little doubt that the reduction in hydrophobicity in nuIMM TMHs is real.

Based on their bioinformatics analysis, the authors also propose that aliphatic -> Thr substitutions (ATS) are the main driver of hydrophobicity reduction in nuIMMs, TMHs, and that Thr has been enriched also in mtIMMs. TMHs in organisms that depend only on Oxa1-type IMM insertases, possibly in order to reduce the aggregation propensity of these proteins when produced in the mitochondrial matrix. This is a novel hypothesis that has not been proposed in earlier work.

The authors additionally show that a mitochondrially encoded ATP6 protein can be converted into a nuclearly encoded, import-competent protein using ATS to reduce the hydrophobicity of its TMHs and, conversely, that import of a nuclearly encoded ATP6 protein is blocked when Thr residues in its TMHs are mutated to aliphatic residues. Similar findings have been published before; what is new is the use of aliphatic -> Thr substitutions (rather than other aliphatic -> polar substitutions).

Detailed comments:

While previous bioinformatics studies of how gene relocation from the mitochondrial to the nuclear genome impacts protein sequence have focused on TMH hydrophobicity, Yadav et al. expand this to include individual amino acid frequencies. The authors present four types of analyses:

(1) The authors identify E. coli homologs of the 13 genes retained in human mitochondrial DNA, do structure-based sequence alignments of the homologous pairs, and then compare the individual aligned TMH pairs in terms of hydrophobicity and Thr content. The authors also calculate an amino acid substitution matrix from the aligned TMH pairs. These data are presented in Figure 2 (and supplements).

Hydrophobicity is calculated based using the GRAVY algorithm and the Kyte-Doolittle scale, and by calculating the fraction of ALIVF residues. Both nuIMM and mtIMM TMHs are found to be significantly less hydrophobic than the E. coli TMHs (p ≈ 0.002). Thr is reported to be enriched in nuIMM and mtIMM TMHs relative to E. coli TMHs (as are Asn and Pro, while Val and Phe are reduced relative to E. coli TMHs, panel C). Panels F and G show an enrichment of Thr residues in nuIMMs of marginal significance (p = 0.02), and an enrichment in mtIMMs of higher significance (but the statistics are skewed by one mtIMM TMH that apparently contains almost 40% Thr residues). The substitution matrix in panel H indicates that aliphatic I, L, A, and G residues in the E. coli TMHs are often substituted by Thr and Val in the mitochondrial proteins. F does not show this substitution pattern, questioning the use of ALIVF counts in the analysis.

(2) The authors calculate hydrophobicities and amino acid frequencies for all human TMHs found in AlphaFold2 structures and grouped according to their cellular location: ER, Golgi, plasma membrane, nucleus, outer mitochondrial membrane (OMM), nuclearly encoded proteins in the inner mitochondrial membrane (nuIMM), and mitochondrially encoded proteins in the inner mitochondrial membrane (mtIMM). These data are presented in Figure 3 (and supplements).

OMM and nuIMM TMHs are significantly less hydrophobic than the secretory pathway TMHs, whereas mtIMM TMHs seem to have similar hydrophobicities as the secretory ones. nuIMM TMHs may have a slightly increased Thr content compared to secretory pathway proteins, but only one p-value is significant (Golgi vs nuIMM). mtIMM TMHs appear to have a higher Thr content than nuIMM proteins, but the statistics is skewed by a single point at a Thr fraction of 0.25. The Thr enrichment is less pronounced than for the E. coli comparison in Figure 2, Figure 3 Supplement. Fig. 8 shows that TMHs in chloroplast and mitochondrial plant proteins both have reduced hydrophobicity compared to ER-targeted proteins.

(3) Starting from the 66 mitochondrially encoded proteins in Jakobids, the authors identify homologous proteins that have become nuclearly encoded and carry an N-terminal mitochondrial targeting sequence (MTS) in organisms with smaller mitochondrial genomes. As in 1, the authors then do structure-based sequence alignments of the 63 homologous nuIMM and mtIMM TMH pairs found, and compare the individual aligned TMH pairs in terms of hydrophobicity and Thr content. These data are presented in Figure 4 (and supplements).

The reduction in hydrophobicity in nuIMM vs mtIMM TMHs upon relocation to the nucleus is robust and confirms earlier publications (panel C). The increase in Thr content in nuIMM vs mtIMM TMHs is less convincing (p = 0.012, panel G) and is opposite to what is seen in Figure 3C.

(4) By phylogenetic analysis, the authors identify three independent relocations of the ATP6 gene to the nuclear genome, do structure-based sequence alignments of nuIMM and mtIMM pairs representing these three events, and illustrate the reduced hydrophobicity of the TMHs in the nuclearly encoded proteins by hydrophobicity plots. These data are presented in Figure 5 (and supplements).

In panel C, it is not only aliphatic-to-Thr substitutions that reduce the hydrophobicity of the nuATP6 TMHs. How much is the hydrophobicity reduced if the Thr mutations are reversed (i.e., how much does the hydrophobicity reduction depend on ATS specifically and not on other kinds of substitutions)?

In a short experimental section, the authors go on to show that a mitochondrially encoded ATP6 protein can be converted into a nuclearly encoded, import-competent protein using ATS to reduce the hydrophobicity of its TMHs (but functionality is not tested) and, conversely, that import of a nuclearly encoded ATP6 protein is blocked when Thr residues in its TMHs are mutated to aliphatic residues (Figures 6, 7).

Finally, the authors carry out all-atom MD simulations to study the energetics of H-bond formation between Thr side chains and TMH backbone atoms.

In summary, the reduction in TMH hydrophobicity in nuIMMs compared to mtIMMs (and to secretory pathway membrane proteins) is robust and nicely confirms earlier studies. The purported enrichment in Thr content in both nuIMMs and mtIMMs has weaker statistical support in the data: p-values are typically > 0.01 (or may be skewed by an outlier) and appear not to have been corrected for multiple testing in all cases. As is clear from the ATP6 data in Figure 5, aliphatic -> Thr substitutions explain only a fraction of the overall hydrophobicity reduction in nuIMM TMHs, and the role of other hydrophobicity-reducing mutations in gene relocation could be better defined.

Author response:

We thank the reviewers for their careful reading of the manuscript and for their insightful comments. Several suggestions relate to a lack of clarity in our presentation, and others propose analyses that will strengthen the impact and robustness of the work. We outline our planned revisions below.

Reviewer 1:

We are grateful to the reviewer for appreciating the significance of our work for the treatment of mitochondrial disorders and its future implications in the development of genetic tools and protein design.

(1) Multiple testing

Figure 3A–C, Figure 3—figure supplements 1, 2, 4 and 5, Figure 6G and Figure 7F used one-way ANOVA with Tukey's post-hoc test, in which correction is intrinsic; the p-values shown are already adjusted. Our legends did not state this, and we will include this information in the revised manuscript.

We will apply Benjamini-Hochberg correction across all 400 comparisons, and as individual cells are likely underpowered, will also test aliphatic-to-threonine substitution at the level of residue classes, analogous to the directional analysis in Figure 4.

(2) Experimental versus predicted structures

We thank the reviewer for pointing this out. Our description here was not adequately clear. All structural alignments and compositional analyses used AlphaFold2 models; experimental structure availability was used only as an inclusion criterion for the compartment comparisons. We chose predicted models because experimental structures are frequently incomplete, which would bias residue composition. We will state this more prominently and add the source of the structure to the relevant legends.

(3) Hydrophobicity scale

We used Kyte-Doolittle for comparability with the prior literature on TMH hydrophobicity in relocated mitochondrial genes, and because it is additive by construction, which the mean-hydrophobicity analyses require. The Hessa scale is defined for insertion of a complete segment and carries substantial position dependence (PMID: 15674282). It is used in Figure 8G because that analysis plots single-residue transfer free energies, which is the scale's intended use and which Kyte-Doolittle, being a hydropathy index does not provide. We will state this rationale in the Methods and in the Figure 8G legend.

(4) Bootstrap support in the ATP6 tree

We agree that the low-support nodes warrant discussion, and we should have addressed it in the original text. These nodes (43, 55, 40) concern relationships among Porifera, Ctenophora and Cnidaria, which remain contested in analyses of far larger datasets (PMID: 41686647). The inference of independent relocation does not depend on them: the apicomplexan, chlorophyte and ctenophore sequences are separated by backbone nodes with 100% support. We thank the reviewer for noting that the distinct MTSs in each clade provide independent support, and we will state this explicitly along with an acknowledgement that values below 70 indicate unresolved relationships.

(5) Core versus peripheral definition

We sincerely thank the reviewer for pointing this out as our text was unclear regarding this. This was set using the ER TMHs, which provide the largest and best-powered set, and applied unchanged to all other groups. The window was chosen to give comparable numbers of positions in each bin, and so that core positions remain membrane-embedded despite helix tilt and uncertainty in the inferred helix midpoint, both of which are greatest near the termini. We will make the rationale explicit and add a robustness analysis at alternative window sizes, as recommended by the reviewer.

(6) Literature

We thank the reviewer for these references and agree they should be discussed. We will incorporate the role of Ser and Thr -containing motifs in TM helix association into the discussion of the biophysical properties of these residues. We will review relevant literature regarding hydrophobicity preferences of the TIM translocon in the context of competition between Sec61 and TIM/TOM insertion, together with the differences between insertion of proteins encoded in the mitochondrial and nuclear genomes. Further, we will also make a version of Figure 8G using the Bothelo and the Kyte-Doolittle scale.

Reviewer 2:

We sincerely appreciate the reviewer for their insightful comments, and for suggesting experiments to further strengthen the manuscript.

(1) Assembly and function of allotopically expressed ATP6

We agree that mitochondrial localization does not establish functional allotopic expression, and will revise the text so this is unambiguous in our manuscript. Our figures 6 and 7 test whether reduced TMH hydrophobicity allows a mitochondrially encoded protein to reach mitochondria when expressed from the nucleus. Assembly into complex V requires further constraints — correct topology, coordinated assembly with the c-ring, and stoichiometry with nuclear-encoded subunits — for which ATS may not be sufficient.

Following the reviewer's thoughtful suggestion, we will perform Blue Native PAGE to test whether the imported subunit is incorporated into ATP synthase and to compare the T1, T2 and T3 constructs. We will also add a Discussion section on topology as a distinct barrier and acknowledge the previous efforts at ATP6 allotopic expression.  As the reviewer anticipates, we expect that it can be challenging for the current constructs to be fully functional, but we will test this experimentally following the BN-PAGE assays. If we do not get successful incorporation, we will discuss this in the text as to what caveats may have caused this.

(2) Expression level confound

We appreciate with the reviewer’s concern regarding this panel. For context, we would like to point out that the conditions originally shown were chosen so that the comparison runs against our conclusion: the most hydrophobic constructs were expressed at low levels and the ATS constructs at high levels, so expression would be expected to work against the observed difference rather than produce it. We recognize that this rationale was not apparent from the figure.

To address this concern further, we have the remaining conditions in hand and will show all four constructs at both expression levels, as requested.

Reviewer 3:

We are very grateful to the reviewer for their thorough review of the manuscript and for providing constructive feedback.

(1) Statistical support for threonine enrichment

We take this point. The strength of statistical support varies across these comparisons, and we will report the p-values with the outliers removed, together with the corrections described under Reviewer 1 point 1. We note that the comparisons are constrained by the number of proteins available there are 13 mtDNA-encoded human IMM proteins, and the E. coli-human comparisons are limited to pairs with detectable homology.

(2) Enrichment of threonine relative to other residues

We will report the absolute residue compositions for the E. coli and human sets in a supplementary figure, and examine the relative enrichment of threonine, asparagine and proline more closely.

(3) Threonine content in Figure 3

The values in Figure 3C are Tukey-adjusted, and all pairwise comparisons are shown in Figure 3—figure supplement 1. The reviewer is correct that only the Golgi comparison reaches significance. Figure 2 compares each human TMH to its aligned E. coli counterpart, so each protein serves as its own control; Figure 3 compares means across unrelated proteins, where between-protein variation is large relative to the compositional difference. We will make this distinction clearer in the text.

(4) Hydrophobicity of mtDNA-encoded TMHs in Figure 3

The reviewer is correct that mtDNA-encoded IMM TMHs do not differ significantly in hydrophobicity from secretory-pathway TMHs. The compartment comparisons test proteins of shared nuclear origin with different destinations; the mtDNA-encoded group is shown for reference, as it differs in both genomic origin and biogenesis route. We will revise the text to specify which mitochondrial class the differences apply to.

(5) Direction of the Figure 3C and Figure 4G comparisons

We thank the reviewer for bringing this up. Our current text did not make it clear that these comparisons involved different organisms. The mtDNA-encoded proteins in Figure 4 are drawn largely from jakobids with large mitochondrial genomes, which retain extensive IMM biogenesis machinery and may face weaker constraints on TMH composition. Those in Figure 3C are the 13 human mitochondrially encoded proteins, which do face these constraints and are correspondingly threonine-rich. The apparent difference in direction reflects this rather than an inconsistency in the underlying trend, and we will make it explicit in the text.

(6) Contribution of ATS to hydrophobicity reduction

We are grateful to the reviewer for their insightful reading of potential evolutionary solutions to the targeting problem. We agree that ATS accounts for part rather than all of the reduction, and that the role of other hydrophobicity-reducing substitutions should be better defined. We will quantify the share attributable to aliphatic-to-threonine substitution, and for the ATP6 examples, report how much of the reduction remains when the threonine substitutions are reverted.

(7) ALIVF grouping

The reviewer is right for noting that phenylalanine does not show substitution towards threonine in the way the other aliphatic residues do in Figure 2H. Phenylalanine is nonetheless depleted in human mitochondrial TMHs alongside isoleucine, valine and alanine (Figure 2C), so the grouping is consistent at the level of composition even though the substitution routes differ. We will also discuss aliphatic-to-valine substitutions, which are largely hydrophobicity-neutral and so do not contribute to the reduction in the same way.

(8) Literature

We thank the reviewer for pointing out early attempts at understanding mitochondrial protein biogenesis and allotopic expression. We will incorporate these references into our discussion, specifically in the context of considering factors which could favor TIM/TOM over Sec61 insertion.