Mitochondrial fusion requires the sequential merger of four bilayers to two. The outer-membrane solute carrier protein SLC25A46 interacts with both the outer and inner-membrane dynamin family GTPases Mfn1/2 and Opa1. While SLC25A46 levels are known to affect mitochondrial morphology, how SLC25A46 interacts with Mfn1/2 and Opa1 to regulate membrane fusion is not understood. In this study, we use crosslinking mass-spectrometry and AlphaFold 2 modeling to identify interfaces mediating a SLC25A46 interactions with Opa1 and Mfn2. We reveal that the bundle signaling element of Opa1 interacts with SLC25A46, and present evidence of a Mfn2 interaction involving the SLC25A46 cytosolic face. We validate these newly identified interaction interfaces and show that they play a role in mitochondrial network maintenance.

Mitochondrial fusion requires the sequential merger of four bilayers to two. The outer-membrane solute carrier protein SLC25A46 interacts with both the outer and inner-membrane dynamin family GTPases Mfn1/2 and Opa1. While SLC25A46 levels are known to affect mitochondrial morphology, how SLC25A46 interacts with Mfn1/2 and Opa1 to regulate membrane fusion is not understood. In this study, we use crosslinking mass-spectrometry and AlphaFold 2 modeling to identify interfaces mediating a SLC25A46 interactions with Opa1 and Mfn2. We reveal that the bundle signaling element of Opa1 interacts with SLC25A46, and present evidence of a Mfn2 interaction involving the SLC25A46 cytosolic face. We validate these newly identified interaction interfaces and show that they play a role in mitochondrial network maintenance.

SLC25A36 is a pyrimidine nucleotide carrier playing an important role in maintaining mitochondrial biogenesis. Deficiencies in SLC25A36 in mouse embryonic stem cells have been associated with mtDNA depletion as well as mitochondrial dysfunction. In human beings, diseases triggered by SLC25A36 mutations have not been described yet. We report the first known case of SLC25A36 deficiency in a 12-year-old patient with hypothyroidism, hyperinsulinism, hyperammonemia, chronical obstipation, short stature, along with language and general developmental delay. Whole exome analysis identified the homozygous mutation c.803dupT, p.Ser269llefs*35 in the SLC25A36 gene. Functional analysis of mutant SLC25A36 protein in proteoliposomes showed a virtually abolished transport activity. Immunoblotting results suggest that the mutant SLC25A36 protein in the patient undergoes fast degradation. Supplementation with oral uridine led to an improvement of thyroid function and obstipation, increase of growth and developmental progress. Our findings suggest an important role of SLC25A36 in hormonal regulations and oral uridine as a safe and effective treatment.

The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate \"revertants\" viable on the glycerol plate by expressing randomly mutated ΔNrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of ΔNrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of ΔNhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria.

Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1(+/gt);Irp1(-/-) erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5\'-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1(gt/gt) cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1(gt/gt) cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.