ABSTRACT
Friedreich's ataxia (FA) is a debilitating, multisystemic disease caused by the depletion of frataxin (FXN), a mitochondrial iron-sulfur (Fe-S) cluster biogenesis factor. To understand the cellular pathogenesis of FA, we performed quantitative proteomics in FXN-deficient human cells. Nearly every annotated Fe-S cluster-containing protein was depleted, indicating that as a rule, cluster binding confers stability to Fe-S proteins. We also observed depletion of a small mitoribosomal assembly factor METTL17 and evidence of impaired mitochondrial translation. Using comparative sequence analysis, mutagenesis, biochemistry, and cryoelectron microscopy, we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]2+ cluster required for its stability. METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN-depleted cells. These findings suggest that METTL17 acts as an Fe-S cluster checkpoint, promoting translation of Fe-S cluster-rich oxidative phosphorylation (OXPHOS) proteins only when Fe-S cofactors are replete.
Subject(s)
Friedreich Ataxia , Iron-Sulfur Proteins , Humans , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Cryoelectron Microscopy , Frataxin , Protein Biosynthesis , Mitochondria/genetics , Mitochondria/metabolism , Friedreich Ataxia/metabolism , Methyltransferases/genetics , Methyltransferases/metabolismABSTRACT
Iron-sulfur clusters (ISC) are essential cofactors that participate in electron transfer, environmental sensing, and catalysis. Amongst the most ancient ISC-containing proteins are the ferredoxin (FDX) family of electron carriers. Humans have two FDXs- FDX1 and FDX2, both of which are localized to mitochondria, and the latter of which is itself important for ISC synthesis. We have previously shown that hypoxia can eliminate the requirement for some components of the ISC biosynthetic pathway, but FDXs were not included in that study. Here, we report that FDX1, but not FDX2, is dispensable under 1% O2 in cultured human cells. We find that FDX1 is essential for production of the lipoic acid cofactor, which is synthesized by the ISC-containing enzyme lipoyl synthase. While hypoxia can rescue the growth phenotype of either FDX1 or lipoyl synthase KO cells, lipoylation in these same cells is not rescued, arguing against an alternative biosynthetic route or salvage pathway for lipoate in hypoxia. Our work reveals the divergent roles of FDX1 and FDX2 in mitochondria, identifies a role for FDX1 in lipoate synthesis, and suggests that loss of lipoic acid can be tolerated under low oxygen tensions in cell culture.
Subject(s)
Ferredoxins , Lipoylation , Humans , Ferredoxins/genetics , Ferredoxins/metabolism , Thioctic Acid/metabolism , Cell Hypoxia/drug effects , Gene Knockout Techniques , Oxygen/pharmacology , Proteome/drug effects , Proteome/genetics , Sulfurtransferases/genetics , Sulfurtransferases/metabolism , Binding Sites , Protein Stability , Protein Biosynthesis/drug effectsABSTRACT
The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.
Subject(s)
Databases, Protein , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Molecular Sequence Annotation , Proteome/metabolism , Animals , Bayes Theorem , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Datasets as Topic , Humans , Internet , Machine Learning , Mass Spectrometry , Mice , Mitochondria/genetics , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/classification , Mitochondrial Proteins/genetics , Proteome/classification , Proteome/genetics , SoftwareABSTRACT
Cellular differentiation involves remodeling cellular architecture to transform one cell type to another. By investigating mitochondrial dynamics during meiotic differentiation in budding yeast, we sought to understand how organelle morphogenesis is developmentally controlled in a system where regulators of differentiation and organelle architecture are known, but the interface between them remains unexplored. We analyzed the regulation of mitochondrial detachment from the cell cortex, a known meiotic alteration to mitochondrial morphology. We found that mitochondrial detachment is enabled by the programmed destruction of the mitochondria-endoplasmic reticulum-cortex anchor (MECA), an organelle tether that bridges mitochondria and the plasma membrane. MECA regulation is governed by a meiotic transcription factor, Ndt80, which promotes the activation of a conserved kinase, Ime2. We further present evidence for Ime2-dependent phosphorylation and degradation of MECA in a temporally controlled manner. Our study defines a key mechanism that coordinates mitochondrial morphogenesis with the landmark events of meiosis and demonstrates that cells can developmentally regulate tethering to induce organelle remodeling.
