SUMMARY Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal new functions for these proteins (termed MXPs) we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend new mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function.

SUMMARY The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. Here, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flips this coenzyme selectivity and enables autophosphorylation, but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.

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.

Background: Lysine acetylation, a prevalent post-translational modification, alters mitochondrial metabolism in response to nutrient changes. Results: Quantitative proteomics distinguishes dynamic and static acetylation sites, highlighting 48 likely regulatory sites of thousands identified. Conclusion: Acetylation of Acat1 lysine 260, a highly dynamic site, reversibly inhibits enzyme activity. Significance: Quantitative, state-specific proteomic analyses accelerate the functional characterization of acetylation in mitochondrial remodeling.

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Summary Deficiency of the serine hydrolase prolyl endopeptidase-like (PREPL) causes a recessive metabolic disorder characterized by neonatal hypotonia, feeding difficulties, and growth hormone deficiency. The pathophysiology of PREPL deficiency and the physiological substrates of PREPL remain largely unknown. In this study, we connect PREPL with mitochondrial gene expression and oxidative phosphorylation by analyzing its protein interactors. We demonstrate that the long PREPL L isoform localizes to mitochondria, whereas PREPL S remains cytosolic. Prepl KO mice showed reduced mitochondrial complex activities and disrupted mitochondrial gene expression. Furthermore, mitochondrial ultrastructure was abnormal in a PREPL-deficient patient and Prepl KO mice. In addition, we reveal that PREPL has (thio)esterase activity and inhibition of PREPL by Palmostatin M suggests a depalmitoylating function. We subsequently determined the crystal structure of PREPL, thereby providing insight into the mechanism of action. Taken together, PREPL is a (thio)esterase rather than a peptidase and PREPL L is involved in mitochondrial homeostasis.

Defining functions for the full complement of proteins is a grand challenge in the post-genomic era and is essential for our understanding of basic biology and disease pathogenesis. In recent times, this endeavor has benefitted from a combination of modern large-scale and classical reductionist approaches-a process we refer to as ''systems biochemistry''-that helps surmount traditional barriers to the characterization of poorly understood proteins. This strategy is proving to be particularly effective for mitochondria, whose well-defined proteome has enabled comprehensive analyses of the full mitochondrial system that can position understudied proteins for fruitful mechanistic investigations. Recent systems biochemistry approaches have accelerated the identification of new disease-related mitochondrial proteins and of long-sought ''missing'' proteins that fulfill key functions. Collectively, these studies are moving us toward a more complete understanding of mitochondrial activities and providing a molecular framework for the investigation of mitochondrial pathogenesis. Mitochondrial Dark MatterThe state of modern biological science is remarkable: our research is performed at a time when ''omics'' techniques enable us to measure nearly every biomolecule, revolutions in imaging and structural biology enable us to observe subcellular components at striking resolution, and gene editing technologies enable us to manipulate DNA seemingly without restriction. Yet, our ability to measure, observe, and manipulate biological systems has arguably outpaced our fundamental understanding of the basic gene functions that underlie them. Recent analyses have revealed that most research on human genes only concentrates on approximately 2,000 of the~19,000 genes of the human genome (Stoeger et al., 2018), and just 100 genes account for more than one-quarter of the papers tagged by the National Library of Medicine (Dolgin, 2017). Similarly, over 600 yeast proteins and 2,000 human proteins have yet to be assigned any molecular function (Ellens et al., 2017), and even for the most well-studied model organisms such as Escherichia coli, more than a third of the genes have yet to be fully functionalized (Ghatak et al., 2019;Sé vin et al., 2017). Relatedly, it is estimated that more than 1,000 known enzyme activities among the 5,000 entries of the Enzyme Commission (EC) classification still lack an associated protein (Sorokina et al., 2014).The reasons why so many proteins remain poorly characterized are manifold. Many are simply hard to study because they might have redundant functions, lack essential functions under standard laboratory conditions, or have functions that affect multiple cellular processes (Oliver, 1996;Hillenmeyer et al., 2008;Galperin and Koonin, 2010). For others, progress is limited by the lack of tools and reagents (e.g., antibodies and mouse lines) available for more ''popular '' proteins (Edwards et al., 2011). Additionally, this lingering focus on a small set of proteins might be rooted in the assumption that th...

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