RESUMO
ADP-ribosylation is a widespread post-translational modification (PTM) with crucial functions in many cellular processes. Here, we describe an in-depth ADP-ribosylome using our Af1521-based proteomics methodology for comprehensive profiling of ADP-ribosylation sites, by systematically assessing complementary proteolytic digestions and precursor fragmentation through application of electron-transfer higher-energy collisional dissociation (EThcD) and electron transfer dissociation (ETD), respectively. Although ETD spectra yielded higher identification scores, EThcD generally proved superior to ETD in identification and localization of ADP-ribosylation sites regardless of protease employed. Notwithstanding, the propensities of complementary proteases and fragmentation methods expanded the detectable repertoire of ADP-ribosylation to an unprecedented depth. This system-wide profiling of the ADP-ribosylome in HeLa cells subjected to DNA damage uncovered >11,000 unique ADP-ribosylated peptides mapping to >7,000 ADP-ribosylation sites, in total modifying over one-third of the human nuclear proteome and highlighting the vast scope of this PTM. High-resolution MS/MS spectra enabled identification of dozens of proteins concomitantly modified by ADP-ribosylation and phosphorylation, revealing a considerable degree of crosstalk on histones. ADP-ribosylation was confidently localized to various amino acid residue types, including less abundantly modified residues, with hundreds of ADP-ribosylation sites pinpointed on histidine, arginine, and tyrosine residues. Functional enrichment analysis suggested modification of these specific residue types is directed in a spatial manner, with tyrosine ADP-ribosylation linked to the ribosome, arginine ADP-ribosylation linked to the endoplasmic reticulum, and histidine ADP-ribosylation linked to the mitochondrion.
Assuntos
ADP-Ribosilação , Espectrometria de Massas/métodos , Proteômica/métodos , Sequência de Aminoácidos , Células HeLa , Humanos , Fosforilação , Proteoma/metabolismo , Serina/metabolismoRESUMO
Despite the importance of citrullination in physiology and disease, global identification of citrullinated proteins, and the precise targeted sites, has remained challenging. Here we employed quantitative-mass-spectrometry-based proteomics to generate a comprehensive atlas of citrullination sites within the HL60 leukemia cell line following differentiation into neutrophil-like cells. We identified 14,056 citrullination sites within 4,008 proteins and quantified their regulation upon inhibition of the citrullinating enzyme PADI4. With this resource, we provide quantitative and site-specific information on thousands of PADI4 substrates, including signature histone marks and transcriptional regulators. Additionally, using peptide microarrays, we demonstrate the potential clinical relevance of certain identified sites, through distinct reactivities of antibodies contained in synovial fluid from anti-CCP-positive and anti-CCP-negative people with rheumatoid arthritis. Collectively, we describe the human citrullinome at a systems-wide level, provide a resource for understanding citrullination at the mechanistic level and link the identified targeted sites to rheumatoid arthritis.
Assuntos
Artrite Reumatoide , Citrulinação , Citrulina , Proteína-Arginina Desiminase do Tipo 4 , Humanos , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Citrulina/metabolismo , Células HL-60 , Proteômica/métodos , Desiminases de Arginina em Proteínas/metabolismo , Desiminases de Arginina em Proteínas/genética , Especificidade por Substrato , Líquido Sinovial/metabolismoRESUMO
ADP-ribosylation is a posttranslational modification (PTM) that has crucial functions in a wide range of cellular processes. Although mass spectrometry (MS) in recent years has emerged as a valuable tool for profiling ADP-ribosylation on a system level, the use of conventional MS methods to profile ADP-ribosylation sites in an unbiased way remains a challenge. Here, we describe a protocol for identification of ADP-ribosylated proteins in vivo on a proteome-wide level, and localization of the amino acid side chains modified with this PTM. The method relies on the enrichment of ADP-ribosylated peptides using the Af1521 macrodomain (Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG, EMBO J 24:1911-1920, 2005), followed by liquid chromatography-high-resolution tandem MS (LC-MS/MS) with electron transfer dissociation-based peptide fragmentation methods, resulting in accurate localization of ADP-ribosylation sites. This protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture to data processing using the MaxQuant software suite.
Assuntos
Adenosina Difosfato Ribose , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Adenosina Difosfato Ribose/química , ADP-Ribosilação , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Peptídeos/químicaRESUMO
Although Poly(ADP-ribose)-polymerases (PARPs) are key regulators of genome stability, how site-specific ADP-ribosylation regulates DNA repair is unclear. Here, we describe a novel role for PARP1 and PARP2 in regulating Rad52-dependent replication fork repair to maintain cell viability when homologous recombination is dysfunctional, suppress replication-associated DNA damage, and maintain genome stability. Mechanistically, Mre11 and ATM are required for induction of PARP activity in response to replication stress that in turn promotes break-induced replication (BIR) through assembly of Rad52 at stalled/damaged replication forks. Further, by mapping ADP-ribosylation sites induced upon replication stress, we identify that PolD3 is a target for PARP1/PARP2 and that its site-specific ADP-ribosylation is required for BIR activity, replication fork recovery and genome stability. Overall, these data identify a critical role for Mre11-dependent PARP activation and site-specific ADP-ribosylation in regulating BIR to maintain genome integrity during DNA synthesis.
Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases , Serina , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , ADP-Ribosilação , Replicação do DNA , Dano ao DNA , Reparo do DNA , Instabilidade GenômicaRESUMO
In the mammalian DNA damage response, ADP-ribosylation signalling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognises damaged DNA and catalyses the formation of serine-linked ADP-ribosylation marks (mono-Ser-ADPr), which are extended into ADP-ribose polymers (poly-Ser-ADPr) by PARP1 alone. Poly-Ser-ADPr is reversed by PARG, while the terminal mono-Ser-ADPr is removed by ARH3. Despite its significance and apparent evolutionary conservation, little is known about ADP-ribosylation signalling in non-mammalian Animalia. The presence of HPF1, but absence of ARH3, in some insect genomes, including Drosophila species, raises questions regarding the existence and reversal of serine-ADP-ribosylation in these species. Here we show by quantitative proteomics that Ser-ADPr is the major form of ADP-ribosylation in the DNA damage response of Drosophila melanogaster and is dependent on the dParp1:dHpf1 complex. Moreover, our structural and biochemical investigations uncover the mechanism of mono-Ser-ADPr removal by Drosophila Parg. Collectively, our data reveal PARP:HPF1-mediated Ser-ADPr as a defining feature of the DDR in Animalia. The striking conservation within this kingdom suggests that organisms that carry only a core set of ADP-ribosyl metabolising enzymes, such as Drosophila, are valuable model organisms to study the physiological role of Ser-ADPr signalling.
Assuntos
Drosophila , Serina , Animais , Serina/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , ADP-Ribosilação , Poli Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Mamíferos/metabolismoRESUMO
The DNA damage response revolves around transmission of information via post-translational modifications, including reversible protein ADP-ribosylation. Here, we applied a mass-spectrometry-based Af1521 enrichment technology for the identification and quantification of ADP-ribosylation sites as a function of various DNA damage stimuli and time. In total, we detected 1681 ADP-ribosylation sites residing on 716 proteins in U2OS cells and determined their temporal dynamics after exposure to the genotoxins H2O2 and MMS. Intriguingly, we observed a widespread but low-abundance serine ADP-ribosylation response at the earliest time point, with later time points centered on increased modification of the same sites. This suggests that early serine ADP-ribosylation events may serve as a platform for an integrated signal response. While treatment with H2O2 and MMS induced homogenous ADP-ribosylation responses, we observed temporal differences in the ADP-ribosylation site abundances. Exposure to MMS-induced alkylating stress induced the strongest ADP-ribosylome response after 30 min, prominently modifying proteins involved in RNA processing, whereas in response to H2O2-induced oxidative stress ADP-ribosylation peaked after 60 min, mainly modifying proteins involved in DNA damage pathways. Collectively, the dynamic ADP-ribosylome presented here provides a valuable insight into the temporal cellular regulation of ADP-ribosylation in response to DNA damage.
Assuntos
ADP-Ribosilação , Dano ao DNA , ADP-Ribosilação/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Peróxido de Hidrogênio/toxicidade , Metanossulfonato de Metila/toxicidade , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Despite the involvement of Poly(ADP-ribose) polymerase-1 (PARP1) in many important biological pathways, the target residues of PARP1-mediated ADP-ribosylation remain ambiguous. To explicate the ADP-ribosylation regulome, we analyze human cells depleted for key regulators of PARP1 activity, histone PARylation factor 1 (HPF1) and ADP-ribosylhydrolase 3 (ARH3). Using quantitative proteomics, we characterize 1,596 ADP-ribosylation sites, displaying up to 1000-fold regulation across the investigated knockout cells. We find that HPF1 and ARH3 inversely and homogenously regulate the serine ADP-ribosylome on a proteome-wide scale with consistent adherence to lysine-serine-motifs, suggesting that targeting is independent of HPF1 and ARH3. Notably, we do not detect an HPF1-dependent target residue switch from serine to glutamate/aspartate under the investigated conditions. Our data support the notion that serine ADP-ribosylation mainly exists as mono-ADP-ribosylation in cells, and reveal a remarkable degree of histone co-modification with serine ADP-ribosylation and other post-translational modifications.
Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas Nucleares/metabolismo , ADP-Ribosilação , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Dano ao DNA , Técnicas de Inativação de Genes , Glicosídeo Hidrolases/genética , Histonas/metabolismo , Humanos , Proteínas Nucleares/genética , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Proteômica , Serina/metabolismoRESUMO
Postsynaptic density protein 95 is a key scaffolding protein in the postsynaptic density of excitatory glutamatergic neurons, organizing signaling complexes primarily via its three PSD-95/Discs-large/Zona occludens domains. PSD-95 is regulated by phosphorylation, but technical challenges have limited studies of the molecular details. Here, we genetically introduced site-specific phosphorylations in single, tandem, and full-length PSD-95 and generated a total of 11 phosphorylated protein variants. We examined how these phosphorylations affected binding to known interaction partners and the impact on phase separation of PSD-95 complexes and identified two new phosphorylation sites with opposing effects. Phosphorylation of Ser78 inhibited phase separation with the glutamate receptor subunit GluN2B and the auxiliary protein stargazin, whereas phosphorylation of Ser116 induced phase separation with stargazin only. Thus, by genetically introducing phosphoserine site-specifically and exploring the impact on phase separation, we have provided new insights into the regulation of PSD-95 by phosphorylation and the dynamics of the PSD.
RESUMO
Poly(ADP-ribose) polymerase 7 (PARP-7) has emerged as a critically important member of a large enzyme family that catalyzes ADP-ribosylation in mammalian cells. PARP-7 is a critical regulator of the innate immune response. What remains unclear is the mechanism by which PARP-7 regulates this process, namely because the protein targets of PARP-7 mono-ADP-ribosylation (MARylation) are largely unknown. Here, we combine chemical genetics, proximity labeling, and proteome-wide amino acid ADP-ribosylation site profiling for identifying the direct targets and sites of PARP-7-mediated MARylation in a cellular context. We found that the inactive PARP family member, PARP-13-a critical regulator of the antiviral innate immune response-is a major target of PARP-7. PARP-13 is preferentially MARylated on cysteine residues in its RNA binding zinc finger domain. Proteome-wide ADP-ribosylation analysis reveals cysteine as a major MARylation acceptor of PARP-7. This study provides insight into PARP-7 targeting and MARylation site preference.
Assuntos
ADP-Ribosilação , Cisteína/metabolismo , Proteínas de Transporte de Nucleosídeos/genética , Proteoma/genética , Proteínas de Ligação a RNA/genética , Mapeamento Cromossômico , Humanos , Proteínas de Transporte de Nucleosídeos/química , Proteoma/química , Proteínas de Ligação a RNA/químicaRESUMO
Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/metabolismo , Nucléolo Celular/genética , DNA Ribossômico/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Motivos de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica , RNA Ribossômico/genética , RNA Ribossômico/metabolismoRESUMO
ADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of activated ion electron transfer dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profile 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress and identify 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and electron-transfer higher-energy collisional dissociation (EThcD), respectively. Under physiological conditions, AI-ETD identifies 450 ADPr sites on low-abundant proteins, including in vivo cysteine modifications on poly(ADP-ribosyl)polymerase (PARP) 8 and tyrosine modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provide insights into the physiological regulation of ADPr.
Assuntos
ADP-Ribosilação/fisiologia , Elétrons , Adenosina Difosfato Ribose/metabolismo , Células HeLa , Humanos , Íons , Poli(ADP-Ribose) Polimerase-1/metabolismoRESUMO
Citrullination, the deimination of peptidylarginine residues into peptidylcitrulline, has been implicated in the etiology of several diseases. In multiple sclerosis, citrullination is thought to be a major driver of pathology through hypercitrullination and destabilization of myelin. As such, inhibition of citrullination has been suggested as a therapeutic strategy for MS. Here, in contrast, we show that citrullination by peptidylarginine deiminase 2 (PAD2) contributes to normal oligodendrocyte differentiation, myelination, and motor function. We identify several targets for PAD2, including myelin and chromatin-related proteins, implicating PAD2 in epigenomic regulation. Accordingly, we observe that PAD2 inhibition and its knockdown affect chromatin accessibility and prevent the upregulation of oligodendrocyte differentiation genes. Moreover, mice lacking PAD2 display motor dysfunction and a decreased number of myelinated axons in the corpus callosum. We conclude that citrullination contributes to proper oligodendrocyte lineage progression and myelination.
Assuntos
Citrulinação , Bainha de Mielina/metabolismo , Oligodendroglia/citologia , Proteína-Arginina Desiminase do Tipo 2/fisiologia , Animais , Diferenciação Celular/genética , Linhagem da Célula , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Perfilação da Expressão Gênica , Camundongos , Oligodendroglia/metabolismo , Mapas de Interação de Proteínas , Proteína-Arginina Desiminase do Tipo 2/análise , Proteína-Arginina Desiminase do Tipo 2/metabolismoRESUMO
ADP-ribosylation (ADPr) is a reversible posttranslational modification involved in a range of cellular processes. Here, we report system-wide identification of serine ADPr in human cells upon oxidative stress. High-resolution mass spectrometry and unrestricted data processing confirm that serine residues are the major target of ADPr in HeLa cells. Proteome-wide analysis identifies 3,090 serine ADPr sites, with 97% of acceptor sites modulating more than 2-fold upon oxidative stress, while treatment with the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib abrogates this induction. Serine ADPr predominantly targets nuclear proteins, while structural-predictive analyses reveal that serine ADPr preferentially targets disordered protein regions. The identified ADP-ribosylated serines significantly overlap with known phosphorylated serines, and large-scale phosphoproteomics analysis provides evidence for site-specific crosstalk between serine ADPr and phosphorylation. Collectively, we demonstrate that serine ADPr is a widespread modification and a major nuclear signaling response to oxidative stress, with a regulatory scope comparable to other extensive posttranslational modifications.
Assuntos
ADP-Ribosilação/fisiologia , Serina/metabolismo , Humanos , FosforilaçãoRESUMO
ADP-ribosylation is a posttranslational modification (PTM) that affects a variety of cellular processes. In recent years, mass spectrometry (MS)-based proteomics has become a valuable tool for studying ADP-ribosylation. However, studying this PTM in vivo in an unbiased and sensitive manner has remained a difficult challenge. Here, we describe a detailed protocol for unbiased analysis of ADP-ribosylated proteins and their ADP-ribose acceptor sites under physiological conditions. The method relies on the enrichment of mono-ADP-ribosylated peptides using the macrodomain Af1521 in combination with liquid chromatography-high-resolution tandem MS (LC-MS/MS). The 5-day protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture stage all the way through to data processing using the MaxQuant software suite.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Humanos , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Proteoma/metabolismo , Proteômica/métodos , SoftwareRESUMO
This study investigates the challenge of comprehensively cataloging the complete human proteome from a single-cell type using mass spectrometry (MS)-based shotgun proteomics. We modify a classical two-dimensional high-resolution reversed-phase peptide fractionation scheme and optimize a protocol that provides sufficient peak capacity to saturate the sequencing speed of modern MS instruments. This strategy enables the deepest proteome of a human single-cell type to date, with the HeLa proteome sequenced to a depth of â¼584,000 unique peptide sequences and â¼14,200 protein isoforms (â¼12,200 protein-coding genes). This depth is comparable with next-generation RNA sequencing and enables the identification of post-translational modifications, including â¼7,000 N-acetylation sites and â¼10,000 phosphorylation sites, without the need for enrichment. We further demonstrate the general applicability and clinical potential of this proteomics strategy by comprehensively quantifying global proteome expression in several different human cancer cell lines and patient tissue samples.
Assuntos
Proteoma/metabolismo , Proteômica/métodos , Células A549 , Acetilação , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas/métodos , Peptídeos/metabolismo , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas/metabolismoRESUMO
The posttranslational modification of proteins by arginine methylation is functionally important, yet the breadth of this modification is not well characterized. Using high-resolution mass spectrometry, we identified 8030 arginine methylation sites within 3300 human proteins in human embryonic kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified by methylation. Through quantitative proteomics and RNA interference to examine arginine methylation stoichiometry, we unexpectedly found that the protein arginine methyltransferase (PRMT) family of arginine methyltransferases catalyzed methylation independently of arginine sequence context. In contrast to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially regulated the functions of the pre-mRNA splicing factor SRSF2 (serine/arginine-rich splicing factor 2) and the RNA transport ribonucleoprotein HNRNPUL1 (heterogeneous nuclear ribonucleoprotein U-like 1). Knocking down PRMT5 impaired the RNA binding function of SRSF2, whereas knocking down PRMT4 [also known as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human arginine methylome provides a missing piece in the global and integrative view of cellular physiology and protein regulation.
Assuntos
Arginina/metabolismo , Neoplasias Ósseas/metabolismo , Osteossarcoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Arginina/química , Células HEK293 , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Metilação , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteômica/métodos , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Although protein ADP-ribosylation is involved in diverse biological processes, it has remained a challenge to identify ADP-ribose acceptor sites. Here, we present an experimental workflow for sensitive and unbiased analysis of endogenous ADP-ribosylation sites, capable of detecting more than 900 modification sites in mammalian cells and mouse liver. In cells, we demonstrate that Lys residues, besides Glu, Asp and Arg residues, are the dominant in vivo targets of ADP-ribosylation during oxidative stress. In normal liver tissue, we find Arg residues to be the predominant modification site. The cellular distribution and biological processes that involve ADP-ribosylated proteins are different in cultured cells and liver tissue, in the latter of which the majority of sites were found to be in cytosolic and mitochondrial protein networks primarily associated with metabolism. Collectively, we describe a robust methodology for the assessment of the role of ADP-ribosylation and ADP-ribosyltransferases in physiological and pathological states.
RESUMO
The essential vitamin biotin is a covalent and tenaciously attached prosthetic group in several carboxylases that play important roles in the regulation of energy metabolism. Here we describe increased acetyl-CoA levels and mitochondrial hyperacetylation as downstream metabolic effects of biotin deficiency. Upregulated mitochondrial acetylation sites correlate with the cellular deficiency of the Hst4p deacetylase, and a biotin-starvation-induced accumulation of Hst4p in mitochondria supports a role for Hst4p in lowering mitochondrial acetylation. We show that biotin starvation and knockout of Hst4p cause alterations in cellular respiration and an increase in reactive oxygen species (ROS). These results suggest that Hst4p plays a pivotal role in biotin metabolism and cellular energy homeostasis, and supports that Hst4p is a functional yeast homologue of the sirtuin deacetylase SIRT3. With biotin deficiency being involved in various metabolic disorders, this study provides valuable insight into the metabolic effects biotin exerts on eukaryotic cells.