TTC19 Plays a Husbandry Role on UQCRFS1 Turnover in the Biogenesis of Mitochondrial Respiratory Complex III.

Loss-of-function mutations in TTC19 (tetra-tricopeptide repeat domain 19) have been associated with severe neurological phenotypes and mitochondrial respiratory chain complex III deficiency. We previously demonstrated the mitochondrial localization of TTC19 and its link with complex III biogenesis. Here we provide detailed insight into the mechanistic role of TTC19, by investigating a Ttc19?/? mouse model that shows progressive neurological and metabolic decline, decreased complex III activity, and increased production of reactive oxygen species. By using both the Ttc19?/? mouse model and a range of human cell lines, we demonstrate that TTC19 binds to the fully assembled complex III dimer, i.e., after the incorporation of the iron-sulfur Rieske protein (UQCRFS1). The in situ maturation of UQCRFS1 produces N-terminal polypeptides, which remain bound to holocomplex III. We show that, in normal conditions, these UQCRFS1 fragments are rapidly removed, but when TTC19 is absent they accumulate within complex III, causing its structural and functional impairment.

Respiratory supercomplexes act as a platform for complex III-mediated maturation of human mitochondrial complexes I and IV.

Mitochondrial respiratory chain (MRC) enzymes associate in supercomplexes (SCs) that are structurally interdependent. This may explain why defects in a single component often produce combined enzyme deficiencies in patients. A case in point is the alleged destabilization of complex I in the absence of complex III. To clarify the structural and functional relationships between complexes, we have used comprehensive proteomic, functional, and biogenetical approaches to analyze a MT-CYB-deficient human cell line. We show that the absence of complex III blocks complex I biogenesis by preventing the incorporation of the NADH module rather than decreasing its stability. In addition, complex IV subunits appeared sequestered within complex III subassemblies, leading to defective complex IV assembly as well. Therefore, we propose that complex III is central for MRC maturation and SC formation. Our results challenge the notion that SC biogenesis requires the pre-formation of fully assembled individual complexes. In contrast, they support a cooperative-assembly model in which the main role of complex III in SCs is to provide a structural and functional platform for the completion of overall MRC biogenesis.

The yeast mitochondrial pyruvate carrier is a hetero-dimer in its functional state.

The mitochondrial pyruvate carrier (MPC) is critical for cellular homeostasis, as it is required in central metabolism for transporting pyruvate from the cytosol into the mitochondrial matrix. MPC has been implicated in many diseases and is being investigated as a drug target. A few years ago, small membrane proteins, called MPC1 and MPC2 in mammals and Mpc1, Mpc2 and Mpc3 in yeast, were proposed to form large protein complexes responsible for this function. However, the MPC complexes have never been isolated and their composition, oligomeric state and functional properties have not been defined. Here, we identify the functional unit of MPC from Saccharomyces cerevisiae In contrast to earlier hypotheses, we demonstrate that MPC is a hetero-dimer, not a multimeric complex. When not engaged in hetero-dimers, the yeast Mpc proteins can also form homo-dimers that are, however, inactive. We show that the earlier described substrate transport properties and inhibitor profiles are embodied by the hetero-dimer. This work provides a foundation for elucidating the structure of the functional complex and the mechanism of substrate transport and inhibition.

METTL15 introduces N4-methylcytidine into human mitochondrial 12S rRNA and is required for mitoribosome biogenesis.

Post-transcriptional RNA modifications, the epitranscriptome, play important roles in modulating the functions of RNA species. Modifications of rRNA are key for ribosome production and function. Identification and characterization of enzymes involved in epitranscriptome shaping is instrumental for the elucidation of the functional roles of specific RNA modifications. Ten modified sites have been thus far identified in the mammalian mitochondrial rRNA. Enzymes responsible for two of these modifications have not been characterized. Here, we identify METTL15, show that it is the main N4-methylcytidine (m4C) methyltransferase in human cells and demonstrate that it is responsible for the methylation of position C839 in mitochondrial 12S rRNA. We show that the lack of METTL15 results in a reduction of the mitochondrial de novo protein synthesis and decreased steady-state levels of protein components of the oxidative phosphorylation system. Without functional METTL15, the assembly of the mitochondrial ribosome is decreased, with the late assembly components being unable to be incorporated efficiently into the small subunit. We speculate that m4C839 is involved in the stabilization of 12S rRNA folding, therefore facilitating the assembly of the mitochondrial small ribosomal subunits. Taken together our data show that METTL15 is a novel protein necessary for efficient translation in human mitochondria.

Stable mitochondrial CICIII2 supercomplex interactions in reptiles versus homeothermic vertebrates.

The association of complex I (CI), complex III (CIII) and complex IV (CIV) of the mitochondrial electron transport chain into stable high molecular weight supercomplexes (SCs) has been observed in several prokaryotes and eukaryotes, but among vertebrates it has only been examined in mammals. The biological role of these SCs is unclear but suggestions so far include enhanced electron transfer between complexes, decreased production of the reactive oxygen species (ROS) O2- and H2O2, or enhanced structural stability. Here, we provide the first overview on the stability, composition and activity of mitochondrial SCs in representative species of several vertebrate classes to determine patterns of SC variation across endotherms and ectotherms. We found that the stability of the CICIII2 SC and the inclusion of CIV within the SC varied considerably. Specifically, when solubilized by the detergent DDM, mitochondrial CICIII2 SCs were unstable in endotherms (birds and mammals) and highly stable in reptiles. Using mass-spectrometric complexomics, we confirmed that the CICIII2 is the major SC in the turtle, and that 90% of CI is found in this highly stable SC. Interestingly, the presence of stable SCs did not prevent mitochondrial H2O2 production and was not associated with elevated respiration rates of mitochondria isolated from the examined species. Together, these data show that SC stability varies among vertebrates and is greatest in poikilothermic reptiles and weakest in endotherms. This pattern suggests an adaptive role of SCs to varying body temperature, but not necessarily a direct effect on electron transfer or in the prevention of ROS production.

Subunit NDUFV3 is present in two distinct isoforms in mammalian complex I.

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the electron transport chain in mammalian mitochondria. Extensive proteomic and structural analyses of complex I from Bos taurus heart mitochondria have shown it comprises 45 subunits encoded on both the nuclear and mitochondrial genomes; 44 of them are different and one is present in two copies. The bovine heart enzyme has provided a model for studying the composition of complex I in other mammalian species, including humans, but the possibility of additional subunits or isoforms in other species or tissues has not been explored. Here, we describe characterization of the complexes I purified from five rat tissues and from a rat hepatoma cell line. We identify a~50kDa isoform of subunit NDUFV3, for which the canonical isoform is only ~10kDa in size. We combine LC-MS and MALDI-TOF mass spectrometry data from two different purification methods (chromatography and immuno-purification) with information from blue native PAGE analyses to show the long isoform is present in the mature complex, but at substoichiometric levels. It is also present in complex I in cultured human cells. We describe evidence that the long isoform is more abundant in both the mitochondria and purified complexes from brain (relative to in heart, liver, kidney and skeletal muscle) and more abundant still in complex I in cultured cells. We propose that the long 50kDa isoform competes with its canonical 10kDa counterpart for a common binding site on the flavoprotein domain of complex I.

The purification and characterization of ATP synthase complexes from the mitochondria of four fungal species.

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-ß-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-ß-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex.

Tapasin-related protein TAPBPR is an additional component of the MHC class I presentation pathway.

Tapasin is an integral component of the peptide-loading complex (PLC) important for efficient peptide loading onto MHC class I molecules. We investigated the function of the tapasin-related protein, TAPBPR. Like tapasin, TAPBPR is widely expressed, IFN-γ-inducible, and binds to MHC class I coupled with ß2-microglobulin in the endoplasmic reticulum. In contrast to tapasin, TAPBPR does not bind ERp57 or calreticulin and is not an integral component of the PLC. ß2-microglobulin is essential for the association between TAPBPR and MHC class I. However, the association between TAPBPR and MHC class I occurs in the absence of a functional PLC, suggesting peptide is not required. Expression of TAPBPR decreases the rate of MHC class I maturation through the secretory pathway and prolongs the association of MHC class I on the PLC. The TAPBPR:MHC class I complex trafficks through the Golgi apparatus, demonstrating a function of TAPBPR beyond the endoplasmic reticulum/cis-Golgi. The identification of TAPBPR as an additional component of the MHC class I antigen-presentation pathway demonstrates that mechanisms controlling MHC class I expression remain incompletely understood.

Endosomal recruitment of the WASH complex: active sequences and mutations impairing interaction with the retromer.

BACKGROUND INFORMATION: The Wiskott-Aldrich syndrome protein and scar homolog (WASH) complex is the major Arp2/3 activator at the surface of endosomes. The branched actin network, that the WASH complex induces, contributes to cargo sorting and scission of transport intermediates destined for most endosomal routes. A major challenge is to understand how the WASH molecular machine is recruited to the surface of endosomes. The retromer endosomal machinery has been proposed by us and others to play a role in this process. RESULTS: In this work, we used an unbiased approach to identify the endosomal receptor of the WASH complex. We have delineated a short fragment of the FAM21 subunit that is able to displace the endogenous WASH complex from endosomes. Using a proteomic approach, we have identified the retromer cargo selective complex (CSC) as a partner of the active FAM21 sequence displacing the endogenous WASH complex. A point mutation in FAM21 that abolishes CSC interaction also impairs WASH complex displacement activity. The CSC is composed of three subunits, VPS35, VPS29 and VPS26. FAM21 directly binds the VPS35 subunit of the retromer CSC. Additionally, we show that a point mutant of VPS35 that blocks binding to VPS29 also prevents association with FAM21 and the WASH complex revealing a novel role for the VPS35-VPS29 interaction in regulating retromer association with the WASH complex. CONCLUSIONS: This novel approach of endogenous WASH displacement confirms previous suggestions that the retromer is the receptor of the WASH complex at the surface of endosomes and identify key residues that mediate this interaction. The interaction between these two endosomal machineries, the WASH complex and the retromer, is likely to play a critical role in forming platforms at the surface of endosomes for efficient sorting of cargoes.

Recruitment of the endosomal WASH complex is mediated by the extended 'tail' of Fam21 binding to the retromer protein Vps35.

The retromer complex is a conserved endosomal protein sorting complex that sorts membrane proteins into nascent endosomal tubules. The recognition of membrane proteins is mediated by the cargo-selective retromer complex, a stable trimer of the Vps35 (vacuolar protein sorting 35), Vps29 and Vps26 proteins. We have recently reported that the cargo-selective retromer complex associates with the WASH (Wiskott-Aldrich syndrome homologue) complex, a multimeric protein complex that regulates tubule dynamics at endosomes. In the present study, we show that the retromer-WASH complex interaction occurs through the long unstructured 'tail' domain of the WASH complex-Fam21 protein binding to Vps35, an interaction that is necessary and sufficient to target the WASH complex to endosomes. The Fam21-tail also binds to FKBP15 (FK506-binding protein 15), a protein associated with ulcerative colitis, to mediate the membrane association of FKBP15. Elevated Fam21-tail expression inhibits the association of the WASH complex with retromer, resulting in increased cytoplasmic WASH complex. Additionally, overexpression of the Fam21-tail results in cell-spreading defects, implicating the activity of the WASH complex in regulating the mobilization of membrane into the endosome-to-cell surface pathway.

Inhibition of METTL3 Results in a Cell-Intrinsic Interferon Response That Enhances Antitumor Immunity.

Therapies that enhance antitumor immunity have altered the natural history of many cancers. Consequently, leveraging nonoverlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyl-transferase METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA (dsRNA) formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signaling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that although METTL3 inhibition is equally efficacious to anti-PD-1 therapy, the combination has far greater preclinical activity. Using SPLINTR barcoding, we demonstrate that anti-PD-1 therapy and METTL3 inhibition target distinct malignant clones, and the combination of these therapies overcomes clones insensitive to the single agents. These data provide the mole-cular and preclinical rationale for employing METTL3 inhibitors to promote antitumor immunity in the clinic. SIGNIFICANCE: This work demonstrates that METTL3 inhibition stimulates a cell-intrinsic interferon response through dsRNA formation. This immunomodulatory mechanism is distinct from current immunotherapeutic agents and provides the molecular rationale for combination with anti-PD-1 immune-checkpoint blockade to augment antitumor immunity. This article is featured in Selected Articles from This Issue, p. 2109.

Evolution of respiratory complex I: "supernumerary" subunits are present in the alpha-proteobacterial enzyme.

Modern α-proteobacteria are thought to be closely related to the ancient symbiont of eukaryotes, an ancestor of mitochondria. Respiratory complex I from α-proteobacteria and mitochondria is well conserved at the level of the 14 "core" subunits, consistent with that notion. Mitochondrial complex I contains the core subunits, present in all species, and up to 31 "supernumerary" subunits, generally thought to have originated only within eukaryotic lineages. However, the full protein composition of an α-proteobacterial complex I has not been established previously. Here, we report the first purification and characterization of complex I from the α-proteobacterium Paracoccus denitrificans. Single particle electron microscopy shows that the complex has a well defined L-shape. Unexpectedly, in addition to the 14 core subunits, the enzyme also contains homologues of three supernumerary mitochondrial subunits as follows: B17.2, AQDQ/18, and 13 kDa (bovine nomenclature). This finding suggests that evolution of complex I via addition of supernumerary or "accessory" subunits started before the original endosymbiotic event that led to the creation of the eukaryotic cell. It also provides further confirmation that α-proteobacteria are the closest extant relatives of mitochondria.

The cargo-selective retromer complex is a recruiting hub for protein complexes that regulate endosomal tubule dynamics.

The retromer complex is required for the efficient endosome-to-Golgi retrieval of the CIMPR, sortilin, SORL1, wntless and other physiologically important membrane proteins. Retromer comprises two protein complexes that act together in endosome-to-Golgi retrieval; the cargo-selective complex is a trimer of VPS35, VPS29 and VPS26 that sorts cargo into tubules for retrieval to the Golgi. Tubules are produced by the oligomerization of sorting nexin dimers. Here, we report the identification of five endosomally-localised proteins that modulate tubule formation and are recruited to the membrane via interactions with the cargo-selective retromer complex. One of the retromer-interacting proteins, strumpellin, is mutated in hereditary spastic paraplegia, a progressive length-dependent axonopathy. Here, we show that strumpellin regulates endosomal tubules as part of a protein complex with three other proteins that include WASH1, an actin-nucleating promoting factor. Therefore, in addition to a direct role in endosome-to-Golgi retrieval, the cargo-selective retromer complex also acts as a platform for recruiting physiologically important proteins to endosomal membranes that regulate membrane tubule dynamics.

Spatial and functional relationship of GGAs and AP-1 in Drosophila and HeLa cells.

The GGAs [Golgi-localised, gamma-ear containing, ARF (ADP ribosylation factor)-binding proteins] and the AP-1 (adaptor protein-1) complex are both adaptors for clathrin-mediated intracellular trafficking, but their relationship to each other is unclear. We have used two complementary systems, HeLa cells and Drosophila Dmel2 cells, to investigate GGA and AP-1 function. Immunoelectron microscopy of endogenous AP-1 and GGA in Dmel2 cells shows that they are predominantly associated with distinct clathrin-coated structures. Depletion of either GGA or AP-1 by RNAi does not affect the incorporation of the other adaptor into clathrin-coated vesicles (CCVs), and the cargo protein GFP-LERP (green fluorescent protein-lysosomal enzyme receptor protein) is lost from CCVs only when both adaptors are depleted. Similar results were obtained using HeLa cells treated with siRNA to deplete all three GGAs simultaneously. AP-1 was still incorporated into CCVs after GGA depletion and vice versa, and both needed to be depleted for a robust inhibition of receptor-mediated sorting of lysosomal hydrolases. In contrast, downregulation of major histocompatibility complex (MHC) class I by HIV-1 Nef, which requires AP-1, was not affected by a triple GGA knockdown. Thus, our results indicate that the two adaptors can function independently of each other.

Quantitative density gradient analysis by mass spectrometry (qDGMS) and complexome profiling analysis (ComPrAn) R package for the study of macromolecular complexes.

Many cellular processes involve the participation of large macromolecular assemblies. Understanding their function requires methods allowing to study their dynamic and mechanistic properties. Here we present a method for quantitative analysis of native protein or ribonucleoprotein complexes by mass spectrometry following their separation by density - qDGMS. Mass spectrometric quantitation is enabled through stable isotope labelling with amino acids in cell culture (SILAC). We provide a complete guide, from experimental design to preparation of publication-ready figures, using a purposely-developed R package - ComPrAn. As specific examples, we present the use of sucrose density gradients to inspect the assembly and dynamics of the human mitochondrial ribosome (mitoribosome), its interacting proteins, the small subunit of the cytoplasmic ribosome, cytoplasmic aminoacyl-tRNA synthetase complex and the mitochondrial PDH complex. ComPrAn provides tools for analysis of peptide-level data as well as normalization and clustering tools for protein-level data, dedicated visualization functions and graphical user interface. Although, it has been developed for the analysis of qDGMS samples, it can also be used for other proteomics experiments that involve 2-state labelled samples separated into fractions. We show that qDGMS and ComPrAn can be used to study macromolecular complexes in their native state, accounting for the dynamics inherent to biological systems and benefiting from its proteome-wide quantitative and qualitative capability.

Duplexing complexome profiling with SILAC to study human respiratory chain assembly defects.

Complexome Profiling (CP) combines size separation, by electrophoresis or other means, of native multimeric complexes with protein identification by mass spectrometry (MS). Peptide MS analysis of the multiple fractions in which the sample is separated, results in the creation of protein abundance profiles in function of molecular size, providing a visual output of the assembly status of a group of proteins of interest. Stable isotope labeling by amino acids in cell culture (SILAC) is an established quantitative proteomics technique that allows duplexing in the MS analysis as well as the comparison of relative protein abundances between the samples, which are processed and analyzed together. Combining SILAC and CP permitted the direct comparison of migration and abundance of the proteins present in the mitochondrial respiratory chain complexes in two different samples. This analysis, however, introduced a level of complexity in data processing for which bioinformatic tools had to be developed in order to generate the normalized protein abundance profiles. The advantages and challenges of using of this type of analysis for the characterization of two cell lines carrying pathological variants in MT-CO3 and MT-CYB is reviewed. An additional unpublished example of SILAC-CP of a cell line with an in-frame 18-bp deletion in MT-CYB is presented. In these cells, in contrast to other MT-CYB deficient models, a small proportion of complex III2 is formed and it is found associated with fully assembled complex I. This analysis also revealed a profuse accumulation of assembly intermediates containing complex III subunits UQCR10 and CYC1, as well as a profound early-stage complex IV assembly defect.

The respiratory complexes I from the mitochondria of two Pichia species.

NADH:ubiquinone oxidoreductase (complex I) is an entry point for electrons into the respiratory chain in many eukaryotes. It couples NADH oxidation and ubiquinone reduction to proton translocation across the mitochondrial inner membrane. Because complex I deficiencies occur in a wide range of neuromuscular diseases, including Parkinson's disease, there is a clear need for model eukaryotic systems to facilitate structural, functional and mutational studies. In the present study, we describe the purification and characterization of the complexes I from two yeast species, Pichia pastoris and Pichia angusta. They are obligate aerobes which grow to very high cell densities on simple medium, as yeast-like, spheroidal cells. Both Pichia enzymes catalyse inhibitor-sensitive NADH:ubiquinone oxidoreduction, display EPR spectra which match closely to those from other eukaryotic complexes I, and show patterns characteristic of complex I in SDS/PAGE analysis. Mass spectrometry was used to identify several canonical complex I subunits. Purified P. pastoris complex I has a particularly high specific activity, and incorporating it into liposomes demonstrates that NADH oxidation is coupled to the generation of a protonmotive force. Interestingly, the rate of NADH-induced superoxide production by the Pichia enzymes is more than twice as high as that of the Bos taurus enzyme. Our results both resolve previous disagreement about whether Pichia species encode complex I, furthering understanding of the evolution of complex I within dikarya, and they provide two new, robust and highly active model systems for study of the structure and catalytic mechanism of eukaryotic complexes I.

The F1 -ATPase from Trypanosoma brucei is elaborated by three copies of an additional p18-subunit.

The F-ATPases (also called the F1 Fo -ATPases or ATP synthases) are multi-subunit membrane-bound molecular machines that produce ATP in bacteria and in eukaryotic mitochondria and chloroplasts. The structures and enzymic mechanisms of their F1 -catalytic domains are highly conserved in all species investigated hitherto. However, there is evidence that the F-ATPases from the group of protozoa known as Euglenozoa have novel features. Therefore, we have isolated pure and active F1 -ATPase from the euglenozoan parasite, Trypanosoma brucei, and characterized it. All of the usual eukaryotic subunits (α, ß, γ, δ, and ε) were present in the enzyme, and, in addition, two unique features were detected. First, each of the three α-subunits in the F1 -domain has been cleaved by proteolysis in vivo at two sites eight residues apart, producing two assembled fragments. Second, the T. brucei F1 -ATPase has an additional subunit, called p18, present in three copies per complex. Suppression of expression of p18 affected in vitro growth of both the insect and infectious mammalian forms of T. brucei. It also reduced the levels of monomeric and multimeric F-ATPase complexes and diminished the in vivo hydrolytic activity of the enzyme significantly. These observations imply that p18 plays a role in the assembly of the F1 domain. These unique features of the F1 -ATPase extend the list of special characteristics of the F-ATPase from T. brucei, and also, demonstrate that the architecture of the F1 -ATPase complex is not strictly conserved in eukaryotes.

MR-1S Interacts with PET100 and PET117 in Module-Based Assembly of Human Cytochrome c Oxidase.

The biogenesis of human cytochrome c oxidase (COX) is an intricate process in which three mitochondrial DNA (mtDNA)-encoded core subunits are assembled in a coordinated way with at least 11 nucleus-encoded subunits. Many chaperones shared between yeast and humans are involved in COX assembly. Here, we have used a MT-CO3 mutant cybrid cell line to define the composition of assembly intermediates and identify new human COX assembly factors. Quantitative mass spectrometry analysis led us to modify the assembly model from a sequential pathway to a module-based process. Each module contains one of the three core subunits, together with different ancillary components, including HIGD1A. By the same analysis, we identified the short isoform of the myofibrillogenesis regulator 1 (MR-1S) as a new COX assembly factor, which works with the highly conserved PET100 and PET117 chaperones to assist COX biogenesis in higher eukaryotes.

Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster.

Altering the redox state of cysteine residues on protein surfaces is an important response to environmental challenges. Although aging and fasting alter many redox processes, the role of cysteine residues is uncertain. To address this, we used a redox proteomic technique, oxidative isotope-coded affinity tags (OxICAT), to assess cysteine-residue redox changes in Drosophila melanogaster during aging and fasting. This approach enabled us to simultaneously identify and quantify the redox state of several hundred cysteine residues in vivo. Cysteine residues within young flies had a bimodal distribution with peaks at ∼10% and ∼85% reversibly oxidized. Surprisingly, these cysteine residues did not become more oxidized with age. In contrast, 24 hr of fasting dramatically oxidized cysteine residues that were reduced under fed conditions while also reducing cysteine residues that were initially oxidized. We conclude that fasting, but not aging, dramatically alters cysteine-residue redox status in D. melanogaster.