Ancestral sequence reconstruction of the Mic60 Mitofilin domain reveals residues supporting respiration in yeast

Background:

Mitochondria are vital organelles found in most eukaryotic cells. Perhaps most importantly, they produce ATP through cellular respiration. Enzymes involved in cellular respiration localise to special subcompartments of mitochondria called cristae. Cristae are tubular and lamellar invaginations of the inner membrane that protrude into the matrix. Though it is not very well understood how cristae form on a molecular level, a large protein complex of the inner membrane, named the mitochondrial contact site and organising system (MICOS), is established to play an important role in the process (Mukherjee et al., 2021).

One of the key proteins within the MICOS complex is Mic60. It is the most conserved subunit of the MICOS complex and contributes to shaping energy-generating membranes not only in eukaryotes, but also in alpha proteobacteria (Munoz-Gomez et al., 2015). Mic60 variants from all domains of life contain a C-terminal mitofilin domain (Munoz-Gomez et al., 2015) that is required for proper membrane architecture and for respiratory growth (Korner et al., 2012, Zerbes et al., 2012). It is widely believed that correct crista structure underlies efficient cellular respiration (Cogliati et al., 2016, Quintana-Cabrera et al., 2018). In the latest update to this preprint, Benning, Bell, Nguyen and team challenge this view by finding mitofilin variants that rescue only respiratory growth, but not cristae structure. Furthermore, by employing ancestral sequence reconstruction of the mitofilin domain, the authors are the first to expose functional molecular differences between Mic60 variants from different domains of life.

 

Key Findings:

1. The C-terminal mitofilin domain of Mic60 is conserved beyond previously defined boundaries

• • • The authors have aligned AlphaFold2-predicted structures of Mic60 from humans, yeast and alphaproteobacteria, and found that a larger part of the C-terminus is structurally conserved than previously thought, comprising a second N-terminal helix bundle, in addition to the already acknowledged C-terminal helix bundle. Therefore, both helix bundles should be considered as comprising the mitofilin domain.

2. Elements of the mitofilin domain that support respiratory growth are not conserved well enough across different domains of life to substitute for each other’s function

• • • Deletion of the mitofilin domain in the budding yeast Saccharomyces cerevisiae ( cerevisiae) leads to a growth deficit on non-fermentable medium, indicating that the domain is required for mitochondrial respiration. The authors tried to rescue respiratory growth by fusing the truncated endogenous S. cerevisiae Mic60 with mitofilin domains from different yeast species, alpha proteobacteria or humans. Only mitofilin domains from yeast could rescue the growth deficit.

3. Yeast-derived ancestors of the mitofilin domain can rescue respiration, but human-derived ancestors cannot

• • • Bioinformatic ancestral sequence reconstruction could “resurrect” the evolutionary ancestors of the mitofilin domain going back to the last common ancestor shared between yeast and humans. The sequence of this last common ancestor, however, ended up to be different depending on whether cerevisiae or Homo sapiens (H. sapiens) was taken as the starting point
    • When the cerevisiae mitofilin domain was swapped for the ancestral mitofilin domain derived from H. sapiens, cellular respiration was as bad as when the mitofilin domain was deleted.

4. Four differentially conserved residues of the mitofilin domain could be found in the yeast and human lineage using multiple sequence alignments. Mutating only these four residues from the yeast amino acids to the human amino acids led to impaired respiratory growth in yeast.

5. Proper crista architecture does not seem to be required for efficient cellular respiration in yeast

• • • Yeast with ancestral mitofilin domains had abberant cristae architecture, resembling that of mitofilin domain deletion mutants. Despite this, the yeast was surprisingly capable of efficient respiratory growth, like wild type yeast.
    • Over half of the mitochondria in yeast, in which the four residues required for efficient respiration were mutated to human-like residues, still possessed attached cristae.

Why I like this preprint:

In my research, I am myself interested in the molecular details of the MICOS complex. One of the biggest challenges in the field, in my eyes, is the multifunctionality of MICOS proteins. I think that finding elements of Mic60 that separate its respiration-supporting function from its role in cristae maintenance is a big step forward. Also, site-specific mutations that impede Mic60 function have to date been very rare. Therefore, I am very excited by the discovery of four specific residues within the Mic60 mitofilin domain whose role may convey species-specific functions of the protein.

Questions/ comments to the authors:

1. Do you expect that mutation of the four residues in human cells to the yeast equivalent amino acids would change the morphology and oxygen consumption rates of human mitochondria?
2. Is it possible to find residues that specifically impede crista junction formation, but not respiration, through multiple alignment of the cerevisiae mitofilin sequence and sequences of yeast-derived mitofilin ancestor strains?
3. Could you expand a little bit more on how you interpret the phenotypes seen in the four-point mutant, as in this particular strain respiratory deficiency and aberrant crista morphology actually seem to correlate to a large extent.

References

COGLIATI, S., ENRIQUEZ, J. A. & SCORRANO, L. 2016. Mitochondrial Cristae: Where Beauty Meets Functionality. Trends Biochem Sci, 41, 261-273.

KORNER, C., BARRERA, M., DUKANOVIC, J., EYDT, K., HARNER, M., RABL, R., VOGEL, F., RAPAPORT, D., NEUPERT, W. & REICHERT, A. S. 2012. The C-terminal domain of Fcj1 is required for formation of crista junctions and interacts with the TOB/SAM complex in mitochondria. Mol Biol Cell, 23, 2143-55.

MUKHERJEE, I., GHOSH, M. & MEINECKE, M. 2021. MICOS and the mitochondrial inner membrane morphology – when things get out of shape. FEBS Lett, 595, 1159-1183.

MUNOZ-GOMEZ, S. A., SLAMOVITS, C. H., DACKS, J. B., BAIER, K. A., SPENCER, K. D. & WIDEMAN, J. G. 2015. Ancient homology of the mitochondrial contact site and cristae organizing system points to an endosymbiotic origin of mitochondrial cristae. Curr Biol, 25, 1489-95.

QUINTANA-CABRERA, R., MEHROTRA, A., RIGONI, G. & SORIANO, M. E. 2018. Who and how in the regulation of mitochondrial cristae shape and function. Biochem Biophys Res Commun, 500, 94-101.

ZERBES, R. M., BOHNERT, M., STROUD, D. A., VON DER MALSBURG, K., KRAM, A., OELJEKLAUS, S., WARSCHEID, B., BECKER, T., WIEDEMANN, N., VEENHUIS, M., VAN DER KLEI, I. J., PFANNER, N. & VAN DER LAAN, M. 2012. Role of MINOS in mitochondrial membrane architecture: cristae morphology and outer membrane interactions differentially depend on mitofilin domains. J Mol Biol, 422,183-91.

 

doi: https://doi.org/10.1242/prelights.39809

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