The extensive and functionally uncharacterized mitochondrial phosphoproteome

Abstract

More than half a century ago, reversible protein phosphorylation was linked to mitochondrial metabolism through the regulation of pyruvate dehydrogenase. Since this discovery, the number of identified mitochondrial protein phosphorylation sites has increased by orders of magnitude, driven largely by technological advances in mass spectrometry-based phosphoproteomics. However, the majority of these modifications remain uncharacterized, rendering their function and relevance unclear. Nonetheless, recent studies have shown that disruption of resident mitochondrial protein phosphatases causes substantial metabolic dysfunction across organisms, suggesting that proper management of mitochondrial phosphorylation is vital for organellar and organismal homeostasis. While these data suggest that phosphorylation within mitochondria is of critical importance, significant gaps remain in our knowledge of how these modifications influence organellar function. Here, we curate publicly available datasets to map the extent of protein phosphorylation within mammalian mitochondria and to highlight the known functions of mitochondrial-resident phosphatases. We further propose models by which phosphorylation may affect mitochondrial enzyme activities, protein import and processing, and overall organellar homeostasis. More than half a century ago, reversible protein phosphorylation was linked to mitochondrial metabolism through the regulation of pyruvate dehydrogenase. Since this discovery, the number of identified mitochondrial protein phosphorylation sites has increased by orders of magnitude, driven largely by technological advances in mass spectrometry-based phosphoproteomics. However, the majority of these modifications remain uncharacterized, rendering their function and relevance unclear. Nonetheless, recent studies have shown that disruption of resident mitochondrial protein phosphatases causes substantial metabolic dysfunction across organisms, suggesting that proper management of mitochondrial phosphorylation is vital for organellar and organismal homeostasis. While these data suggest that phosphorylation within mitochondria is of critical importance, significant gaps remain in our knowledge of how these modifications influence organellar function. Here, we curate publicly available datasets to map the extent of protein phosphorylation within mammalian mitochondria and to highlight the known functions of mitochondrial-resident phosphatases. We further propose models by which phosphorylation may affect mitochondrial enzyme activities, protein import and processing, and overall organellar homeostasis. Metabolism must respond to dynamic shifts in nutrient availability and energy demands. Phosphorylation, being a rapid and reversible posttranslational modification (PTM), is well suited to calibrate these needs. Indeed, phosphorylation has long been known to modulate metabolic signaling: the first protein associated with phosphorylation-based regulation was glycogen phosphorylase (1Fischer E.H. Krebs E.G. Conversion of phosphorylase b to phosphorylase a in muscle extracts.J. Biol. Chem. 1955; 216: 121-132Abstract Full Text PDF PubMed Google Scholar)—a discovery first reported in 1955 by Edwin Krebs and Edmond Fischer that earned them the 1992 Nobel Prize in Physiology and Medicine (2Cohen P. The origins of protein phosphorylation.Nat. Cell Biol. 2002; 4: E127-130Crossref PubMed Scopus (724) Google Scholar). Early observations also linked phosphorylation to mitochondria. Albert Lehninger's group noted in the mid-1940s that incorporation of radioactive phosphate into rat liver phosphoproteins was dependent upon oxidative phosphorylation (3Friedkin M. Lehninger A.L. Oxidation-coupled incorporation of inorganic radiophosphate into phospholipide and nucleic acid in a cell-free system.J. Biol. Chem. 1949; 177: 775-788Abstract Full Text PDF PubMed Google Scholar), and Eugene Kennedy and colleagues first demonstrated cellular kinase activity against casein using soluble mitochondrial extract in 1954 (4Burnett G. Kennedy E.P. The enzymatic phosphorylation of proteins.J. Biol. Chem. 1954; 211: 969-980Abstract Full Text PDF PubMed Google Scholar). However, the discovery of pyruvate dehydrogenase (PDH) regulation by phosphorylation in 1969 by Lester Reed's group (and Otto Wieland shortly thereafter) firmly established the role of this PTM in mitochondrial biology (5Linn T.C. Pettit F.H. Reed L.J. Alpha-keto acid dehydrogenase complexes. X. Regulation of the activity of the pyruvate dehydrogenase complex from beef kidney mitochondria by phosphorylation and dephosphorylation.Proc. Natl. Acad. Sci. U. S. A. 1969; 62: 234-241Crossref PubMed Scopus (513) Google Scholar, 6Wieland O. Siess E. Interconversion of phospho- and dephospho- forms of pig heart pyruvate dehydrogenase.Proc. Natl. Acad. Sci. U. S. A. 1970; 65: 947-954Crossref PubMed Scopus (116) Google Scholar). Since these seminal discoveries, major technological advances in mass spectrometry (MS)-based quantitative phosphoproteomics have uncovered tens of thousands of phosphorylation sites on proteins across various cell types, tissues, and organisms (7Hornbeck P.V. Zhang B. Murray B. Kornhauser J.M. Latham V. Skrzypek E. PhosphoSitePlus, 2014: Mutations, PTMs and recalibrations.Nucleic Acids Res. 2015; 43: D512-520Crossref PubMed Scopus (1430) Google Scholar). By curating many of these datasets, we found, perhaps surprisingly, that ∼91% of annotated mitochondrial proteins have at least one reported phosphorylation site as of March 2021 (Fig. 1A). Furthermore, these mitochondrial proteins have an average of approximately eight distinct phosphorylation sites (median = 5, Fig. 1B), demonstrating that this modification is highly prevalent across the mitochondrial proteome. Are these modifications broadly meaningful for mitochondrial biology? The answer has remained unclear for some time. Selecting and biochemically characterizing individual phosphorylation events from these large, complex datasets has proven challenging, obscuring insight into how these modifications might directly alter mitochondrial protein function. Thus, despite their prevalence, less than 5% of these phosphorylated residues are associated with any published investigation (Fig. 1C), and even these few “characterized” modifications often lack critical details regarding function, stoichiometry (i.e., the fraction of phosphorylated relative to unphosphorylated protein), and regulation across physiological contexts. By some measures, it is reasonable to assume that these PTMs are a trivial distraction—biological “noise” captured by instruments with ever-increasing sensitivity. Indeed, systems-wide analyses have found that mitochondrial phosphoproteins typically exhibit low stoichiometry (8Wu R. Haas W. Dephoure N. Huttlin E.L. Zhai B. Sowa M.E. Gygi S.P. A large-scale method to measure absolute protein phosphorylation stoichiometries.Nat. Methods. 2011; 8: 677-683Crossref PubMed Scopus (213) Google Scholar). Low-stoichiometry modifications need not be nonfunctional on principle; modifications that activate enzyme function, for example, can substantially alter biological function at low stoichiometry (9Prus G. Hoegl A. Weinert B.T. Choudhary C. Analysis and interpretation of protein post-translational modification site stoichiometry.Trends Biochem. Sci. 2019; 44: 943-960Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Even for inhibitory modifications, the collective accumulation of low-stoichiometry events could decrease organellar function, as has been shown for mitochondrial protein acylation modifications (10Weinert B.T. Moustafa T. Iesmantavicius V. Zechner R. Choudhary C. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions.EMBO J. 2015; 34: 2620-2632Crossref PubMed Scopus (90) Google Scholar, 11Baeza J. Smallegan M.J. Denu J.M. Mechanisms and dynamics of protein acetylation in mitochondria.Trends Biochem. Sci. 2016; 41: 231-244Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Nonetheless, it is reasonable to assume that low-stoichiometry modifications are less likely to have biological impact and may reflect spurious events. However, other observations suggest that these PTMs may possess important regulatory value. For example, multiple studies have found that phosphorylation sites on mitochondrial proteins are reproducibly dynamic across physiological states and models of disease (12Grimsrud P.A. Carson J.J. Hebert A.S. Hubler S.L. Niemi N.M. Bailey D.J. Jochem A. Stapleton D.S. Keller M.P. Westphall M.S. Yandell B.S. Attie A.D. Coon J.J. Pagliarini D.J. A quantitative map of the liver mitochondrial phosphoproteome reveals posttranslational control of ketogenesis.Cell Metab. 2012; 16: 672-683Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 13Zhao X. Leon I.R. Bak S. Mogensen M. Wrzesinski K. Hojlund K. Jensen O.N. Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes.Mol. Cell Proteomics. 2011; 10 (M110 000299)Abstract Full Text Full Text PDF Scopus (116) Google Scholar, 14Deng N. Zhang J. Zong C. Wang Y. Lu H. Yang P. Wang W. Young G.W. Wang Y. Korge P. Lotz C. Doran P. Liem D.A. Apweiler R. Weiss J.N. et al.Phosphoproteome analysis reveals regulatory sites in major pathways of cardiac mitochondria.Mol. Cell Proteomics. 2011; 10 (M110 000117)Abstract Full Text Full Text PDF Scopus (80) Google Scholar, 15Reinders J. Wagner K. Zahedi R.P. Stojanovski D. Eyrich B. van der Laan M. Rehling P. Sickmann A. Pfanner N. Meisinger C. Profiling phosphoproteins of yeast mitochondria reveals a role of phosphorylation in assembly of the ATP synthase.Mol. Cell Proteomics. 2007; 6: 1896-1906Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), implying functionality (or, at least, nonrandomness). Moreover, the select examples of purified mitochondrial phosphoproteins or phosphomimetics show that these PTMs certainly can alter protein activity when present (albeit, often at stoichiometry higher than what was observed in cells or tissues). Collectively, these observations demand better answers to two fundamental questions: are phosphorylation sites on mitochondrial proteins functionally meaningful? If so, which? In debating the answers to the questions above, we were struck by another key data point: mitochondria appear to possess a set of resident protein phosphatases. The MitoCarta compendia have included 12 candidate protein phosphatases (16Pagliarini D.J. Calvo S.E. Chang B. Sheth S.A. Vafai S.B. Ong S.E. Walford G.A. Sugiana C. Boneh A. Chen W.K. Hill D.E. Vidal M. Evans J.G. Thorburn D.R. Carr S.A. et al.A mitochondrial protein compendium elucidates complex I disease biology.Cell. 2008; 134: 112-123Abstract Full Text Full Text PDF PubMed Scopus (1430) Google Scholar, 17Calvo S.E. Clauser K.R. Mootha V.K. MitoCarta2.0: An updated inventory of mammalian mitochondrial proteins.Nucleic Acids Res. 2016; 44: D1251-1257Crossref PubMed Scopus (791) Google Scholar, 18Rath S. Sharma R. Gupta R. Ast T. Chan C. Durham T.J. Goodman R.P. Grabarek Z. Haas M.E. Hung W.H.W. Joshi P.R. Jourdain A.A. Kim S.H. Kotrys A.V. Lam S.S. et al.MitoCarta3.0: An updated mitochondrial proteome now with sub-organelle localization and pathway annotations.Nucleic Acids Res. 2021; 49: D1541-D1547Crossref PubMed Scopus (128) Google Scholar) (Fig. 2A), many of which are conserved across eukaryotic species, suggesting ancient origins for mitochondrial dephosphorylation (19Frankovsky J. Vozarikova V. Nosek J. Tomaska L. Mitochondrial protein phosphorylation in yeast revisited.Mitochondrion. 2021; 57: 148-162Crossref PubMed Scopus (5) Google Scholar) (Fig. 2B). The presence of 12 mitochondrial phosphatases in a single organelle may, at first glance, imply substantial functional redundancy, but evidence suggests that these proteins have distinct, nonoverlapping roles. First, mitochondrial phosphatases exploit five distinct catalytic domains, each with specific residue preferences that confer substrate selectivity (Fig. 2C). Furthermore, these phosphatases are subcompartmentalized across the organelle (20Hung V. Zou P. Rhee H.W. Udeshi N.D. Cracan V. Svinkina T. Carr S.A. Mootha V.K. Ting A.Y. Proteomic mapping of the human mitochondrial intermembrane space in live cells via ratiometric APEX tagging.Mol. Cell. 2014; 55: 332-341Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 21Hung V. Lam S.S. Udeshi N.D. Svinkina T. Guzman G. Mootha V.K. Carr S.A. Ting A.Y. Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation.Elife. 2017; 6Crossref Scopus (157) Google Scholar, 22Rhee H.W. Zou P. Udeshi N.D. Martell J.D. Mootha V.K. Carr S.A. Ting A.Y. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.Science. 2013; 339: 1328-1331Crossref PubMed Scopus (651) Google Scholar), physically dividing substrate pools and functions. RNA-seq profiles of many mitochondrial phosphatases show tissue-specific expression (23Carithers L.J. Ardlie K. Barcus M. Branton P.A. Britton A. Buia S.A. Compton C.C. DeLuca D.S. Peter-Demchok J. Gelfand E.T. Guan P. Korzeniewski G.E. Lockhart N.C. Rabiner C.A. Rao A.K. et al.A novel approach to high-quality postmortem tissue procurement: The GTEx project.Biopreserv Biobank. 2015; 13: 311-319Crossref PubMed Scopus (387) Google Scholar), further diversifying functions in vivo (Fig. 2D). Finally, genetic or pharmacological perturbation of many mitochondrial phosphatases results in distinct—and often severe—phenotypes, suggesting that these enzymes enable mitochondrial homeostasis through unique functions. Given these insights, our approach to addressing the questions above has been to profile the mitochondrial phosphoproteome following the perturbation of one of these phosphatases (24Guo X. Niemi N.M. Hutchins P.D. Condon S.G.F. Jochem A. Ulbrich A. Higbee A.J. Russell J.D. Senes A. 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The found on many mitochondrial upon to the organelle and is to influence protein function. than of mitochondrial proteins are phosphorylated within or to this (Fig. data suggest that phosphorylation may by to protein import and may influence the mitochondrial import of If phosphorylated mitochondrial can be into mitochondria it can in K. J. of the of proteins.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (155) Google this might to of the observed mitochondrial regulation of mitochondrial as in to various cellular could mitochondrial to affect their organellar and function. could a rapid and reversible to specific within mitochondria dynamic cellular Furthermore, this could two in the of mitochondrial the of relative to mitochondrial and at least for the low are and The has an in the and of which cellular and have the of phosphorylation on mitochondrial However, questions remain regarding which of these modifications influence organellar function and the physiological which proteins phosphorylated within the

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