Mitochondrial dysfunction is associated with many human diseases, including cancer and neurodegeneration, that are often linked to proteins and pathways that are not well-characterized. To begin defining the functions of such poorly characterized proteins, we used mass spectrometry to map the proteomes, lipidomes and metabolomes of 174 yeast strains, each lacking a single gene related to mitochondrial biology. 144 of these genes have human homologs, 60 of which are associated with disease and 39 of which are uncharacterized. We present a multi-omic data analysis and visualization tool that we use to find covariance networks that can predict molecular functions, correlations between profiles of related gene deletions, gene-specific perturbations that reflect protein functions, and a global respiration deficiency response. Using this multi-omic approach, we link seven proteins including Hfd1p and its human homolog ALDH3A1 to mitochondrial coenzyme Q (CoQ) biosynthesis, an essential pathway disrupted in many human diseases. This Resource should provide broad molecular insights into mitochondrial protein functions.

Summary SIRT3 is a member of the Sirtuin family of NAD+-dependent deacylases and plays a critical role in metabolic regulation. Organism-wide SIRT3 loss manifests in metabolic alterations, however the coordinating role of SIRT3 among metabolically distinct tissues is unknown. Using multi-tissue quantitative proteomics comparing fasted wild type mice to mice lacking SIRT3, innovative bioinformatic analysis, and biochemical validation, we provide a comprehensive view of mitochondrial acetylation and SIRT3 function. We find SIRT3 regulates the acetyl-proteome in core mitochondrial processes common to brain, heart, kidney, liver, and skeletal muscle, but differentially regulates metabolic pathways in fuel-producing and fuel-utilizing tissues. We propose an additional maintenance function for SIRT3 in liver and kidney where SIRT3 expression is elevated to reduce the acetate load on mitochondrial proteins. We provide evidence that SIRT3 impacts ketone body utilization in the brain and reveal a pivotal role for SIRT3 in the coordination between tissues required for metabolic homeostasis.

SUMMARY The UbiB protein kinase-like (PKL) family is widespread—comprising one-quarter of microbial PKLs and five human homologs—yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. While COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates—functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small molecules for probing its activity in vivo. Here, we demonstrate that COQ8 possesses evolutionarily conserved ATPase activity that is activated by binding to membranes containing cardiolipin and by phenolic compounds that resemble CoQ pathway intermediates. We further create an analog-sensitive version of Coq8p and reveal that acute chemical inhibition of its endogenous activity in yeast is sufficient to cause respiratory deficiency concomitant with CoQ depletion. Collectively, this work defines lipid and small-molecule modulators of an ancient family of atypical kinase-like proteins and establishes a chemical genetic system for further exploring the mechanistic role of COQ8 in CoQ biosynthesis.

Beyond its role in mitochondrial bioenergetics, Coenzyme Q (CoQ, ubiquinone) serves as a key membrane-embedded antioxidant throughout the cell. However, how CoQ is mobilized from its site of synthesis on the inner mitochondrial membrane to other sites of action remains a longstanding mystery. Here, using a combination of Saccharomyces cerevisiae genetics, biochemical fractionation, and lipid profiling, we identify two highly conserved but poorly characterized mitochondrial proteins, Ypl109c (Cqd1) and Ylr253w (Cqd2), that reciprocally affect this process. Loss of Cqd1 skews cellular CoQ distribution away from mitochondria, resulting in markedly enhanced resistance to oxidative stress caused by exogenous polyunsaturated fatty acids, whereas loss of Cqd2 promotes the opposite effects. The activities of both proteins rely on their atypical kinase/ATPase domains, which they share with Coq8—an essential auxiliary protein for CoQ biosynthesis. Overall, our results reveal protein machinery central to CoQ trafficking in yeast and lend insights into the broader interplay between mitochondria and the rest of the cell.

The Nrf2 transcription factor is a master regulator of the cellular defense against oxidative and electrophilic stress. An increase in Nrf2 protein levels and an accumulation of Nrf2 in the nucleus are key parts of the Nrf2 activation mechanism. The western blot technique remains the most widely used method to assess these changes. A well-characterized, specific antibody that is commercially available would greatly enhance these studies in the field. Here, an apparently highly specific Nrf2 monoclonal antibody, EP1808Y from Abcam, is compared with the most widely used Nrf2 antibodies, H-300 and C-20, both from Santa Cruz Biotechnology, in a panel of human cell lines. In addition to detecting Nrf2, EP1808Y avidly detects another protein present in two of the three cell lines tested. This protein can be mistaken for Nrf2 as it co-migrates with verified Nrf2 on two different polyacrylamide gel types. However, unlike Nrf2, its levels and cytoplasmic localization are unaffected by treatment with Nrf2 activators. The possibility that this band corresponds to a form of Nrf2 was excluded by siRNA and immunodepletion experiments. Finally, the monoclonal antibody D1Z9C from Cell Signaling was found to detect Nrf2 with the highest specificity of these four antibodies.

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SummaryHuman COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small molecules for probing its activity in vivo. Here we demonstrate that COQ8 possesses evolutionarily conserved ATPase activity that is activated by binding to membranes containing cardiolipin and by phenolic compounds that resemble CoQ pathway intermediates. We further create an analog-sensitive version of Coq8p and reveal that acute chemical inhibition of its endogenous activity in yeast is sufficient to cause respiratory deficiency concomitant with CoQ depletion. Collectively, this work defines lipid and small molecule modulators of an ancient family of atypical kinase-like proteins and establishes a chemical genetic system for further exploring the mechanistic role of COQ8 in CoQ biosynthesis.