Mitochondrial copper and phosphate transporter specificity was defined early in the evolution of eukaryotes
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Abstract
The mitochondrial carrier family protein SLC25A3 transports both copper and phosphate in mammals, yet in Saccharomyces cerevisiae the transport of these substrates is partitioned across two paralogs: PIC2 and MIR1. To understand the ancestral state of copper and phosphate transport in mitochondria, we explored the evolutionary relationships of PIC2 and MIR1 orthologs across the eukaryotic tree of life. Phylogenetic analyses revealed that PIC2-like and MIR1-like orthologs are present in all major eukaryotic supergroups, indicating an ancient gene duplication created these paralogs. To link this phylogenetic signal to protein function, we used structural modeling and site-directed mutagenesis to identify residues involved in copper and phosphate transport. Based on these analyses, we generated an L175A variant of mouse SLC25A3 that retains the ability to transport copper but not phosphate. This work highlights the utility of using an evolutionary framework to uncover amino acids involved in substrate recognition by mitochondrial carrier family proteins.
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Summary: This work synthesizes bioinformatics, in vivo, and in vitro transport assays to understand the molecular basis for substrate selection and promiscuity of the mitochondrial carrier family (SLC25). This comprehensive work will be of interest to the fields of mitochondrial physiology, transporter specificity and evolutionary dynamics. However, in its current form, it lacks some critical controls for protein expression and some important details about the methodology.
Reviewer #1 and Reviewer #2 opted to reveal their name to the authors in the decision letter after review.
Public review:
This paper takes a novel and comprehensive approach to understand the molecular basis for substrate selection and promiscuity of the mitochondrial carrier family (SLC25). Informed by a deep assessment of evolutionarily conserved features, mutants …
Summary: This work synthesizes bioinformatics, in vivo, and in vitro transport assays to understand the molecular basis for substrate selection and promiscuity of the mitochondrial carrier family (SLC25). This comprehensive work will be of interest to the fields of mitochondrial physiology, transporter specificity and evolutionary dynamics. However, in its current form, it lacks some critical controls for protein expression and some important details about the methodology.
Reviewer #1 and Reviewer #2 opted to reveal their name to the authors in the decision letter after review.
Public review:
This paper takes a novel and comprehensive approach to understand the molecular basis for substrate selection and promiscuity of the mitochondrial carrier family (SLC25). Informed by a deep assessment of evolutionarily conserved features, mutants that selectively impair Pi flux, but retain Cu2+ transport for the mammalian transporter SLC25A3 were established using a variety of in vitro and in vivo transport assays. In addition to providing a molecular perspective on substrate specificity in mitochondrial carrier proteins, this paper provides interesting and convincing insight into how subfamilies of transporters evolved by juggling substrate specificity. However, in its current form, it lacks some critical controls for protein expression and some important details about the methodology, which are enumerated below:
This manuscript does not report any controls for expression levels or membrane localization of the mutants analyzed in Figure 6. These controls are essential to fairly compare the growth phenotypes/transport capacity of the assorted mutants relative to WT Pic2.
The methods section lacks details required to fully understand several different experiments.
-Figure 1G shows an analysis with reconstituted proteins, but the methods contain no information about purification or reconstitution of the transporters, or the origin of the CuL fluorescent reporter, so it is difficult to evaluate this line of evidence.
-The methods do not contain information about the NMR experiment shown in Figure S4, and the interpretation of this data as containing a benzene ring is probably not obvious to a broader scientific audience.
-The details are also sparse regarding the preparation of the homology model. How much sequence similarity do the ATP/ADP translocase and PIC2 share? How large are the insertions and deletions that were addressed by manual alignment? Was an ensemble of models calculated? It is likely that a number of plausible models could be produced - were any alternative models considered? The clustering of the conserved residues shown in Figure 4 is a nice way to validate the model. It would also be nice to analyze whether the homology model shows the expected pattern of hydrophobic residues facing the membrane.
-The authors should include all details of the bioinformatic pipeline as supplementary data, including the list of gene ids and/or sequences, phylogenetic tree of the initial 2445 sequences (neighbour joining tree) to show PIC2/MIR1 clusters, and the 92 final sequences (gene IDs, multiple sequence alignment). In addition, the authors should show the entire tree of the superfamily.
- The manuscript would be strengthened by additional discussion about what is known (if anything) about the functions of other transporters in the PIC2/MIR1 family. Much of the interpretation of the phylogeny regarding the outcomes of gene duplication seem to depend critically on whether the functions and substrate specificities of the yeast and mammalian homologues described here are representative of the entire clade. Likewise, the authors do not indicate whether there is evidence that neighboring sequences outside the core PIC2/MIR1 cluster are not functionally homologous (promiscuous Cu and/or phosphate transport) to PIC1/MIR1.
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