New aspects of redox signaling mediated by supersulfides in health and disease.

Oxygen molecules accept electrons from the respiratory chain in the mitochondria and are responsible for energy production in aerobic organisms. The reactive oxygen species formed via these oxygen reduction processes undergo complicated electron transfer reactions with other biological substances, which leads to alterations in their physiological functions and cause diverse biological and pathophysiological consequences (e.g., oxidative stress). Oxygen accounts for only a small proportion of the redox reactions in organisms, especially under aerobic or hypoxic conditions but not under anaerobic and hypoxic conditions. This article discusses a completely new concept of redox biology, which is governed by redox-active supersulfides, i.e., sulfur-catenated molecular species. These species are present in abundance in all organisms but remain largely unexplored in terms of redox biology and life science research. In fact, accumulating evidence shows that supersulfides have extensive redox chemical properties and that they can be readily ionized or radicalized to participate in energy metabolism, redox signaling, and oxidative stress responses in cells and in vivo. Thus, pharmacological intervention and medicinal modulation of supersulfide activities have been shown to benefit the regulation of disease pathogenesis as well as disease control.

2H-Thiopyran-2-thione sulfine, a compound for converting H2S to HSOH/H2S2 and increasing intracellular sulfane sulfur levels.

Reactive sulfane sulfur species such as persulfides (RSSH) and H2S2 are important redox regulators and closely linked to H2S signaling. However, the study of these species is still challenging due to their instability, high reactivity, and the lack of suitable donors to produce them. Herein we report a unique compound, 2H-thiopyran-2-thione sulfine (TTS), which can specifically convert H2S to HSOH, and then to H2S2 in the presence of excess H2S. Meanwhile, the reaction product 2H-thiopyran-2-thione (TT) can be oxidized to reform TTS by biological oxidants. The reaction mechanism of TTS is studied experimentally and computationally. TTS can be conjugated to proteins to achieve specific delivery, and the combination of TTS and H2S leads to highly efficient protein persulfidation. When TTS is applied in conjunction with established H2S donors, the corresponding donors of H2S2 (or its equivalents) are obtained. Cell-based studies reveal that TTS can effectively increase intracellular sulfane sulfur levels and compensate for certain aspects of sulfide:quinone oxidoreductase (SQR) deficiency. These properties make TTS a conceptually new strategy for the design of donors of reactive sulfane sulfur species.

Longevity control by supersulfide-mediated mitochondrial respiration and regulation of protein quality.

Supersulfides, which are defined as sulfur species with catenated sulfur atoms, are increasingly being investigated in biology. We recently identified pyridoxal phosphate (PLP)-dependent biosynthesis of cysteine persulfide (CysSSH) and related supersulfides by cysteinyl-tRNA synthetase (CARS). Here, we investigated the physiological role of CysSSH in budding yeast (Saccharomyces cerevisiae) by generating a PLP-binding site mutation K109A in CRS1 (the yeast ortholog of CARS), which decreased the synthesis of CysSSH and related supersulfides and also led to reduced chronological aging, effects that were associated with an increased endoplasmic reticulum stress response and impaired mitochondrial bioenergetics. Reduced chronological aging in the K109A mutant could be rescued by using exogenous supersulfide donors. Our findings indicate important roles for CARS in the production and metabolism of supersulfides-to mediate mitochondrial function and to regulate longevity.

Quantitative profiling of supersulfides naturally occurring in dietary meats and beans.

Sulfur is essential in the inception of life and crucial for maintaining human health. This mineral is primarily supplied through the intake of proteins and is used for synthesizing various sulfur-containing biomolecules. Recent research has highlighted the biological significance of endogenous supersulfides, which include reactive persulfide species and sulfur catenated residues in thiol and proteins. Ingestion of exogenous sulfur compounds is essential for endogenous supersulfide production. However, the content and composition of supersulfides in foods remain unclear. This study investigated the supersulfide profiles of protein-rich foods, including edible animal meat and beans. Quantification of the supersulfide content revealed that natto, chicken liver, and bean sprouts contained abundant supersulfides. In general, the supersulfide content in beans and their derivatives was higher than that in animal meat. The highest proportion (2.15 %) was detected in natto, a traditional Japanese fermented soybean dish. These results suggest that the abundance of supersulfides, especially in foods like natto and bean sprouts, may contribute to their health-promoting properties. Our findings may have significant biological implications and warrant developing novel dietary intervention for the human health-promoting effects of dietary supersulfides abundantly present in protein-rich foods such as natto and bean sprouts.

Supersulfide catalysis for nitric oxide and aldehyde metabolism.

Abundant formation of endogenous supersulfides, which include reactive persulfide species and sulfur catenated residues in thiols and proteins (supersulfidation), has been observed. We found here that supersulfides catalyze S-nitrosoglutathione (GSNO) metabolism via glutathione-dependent electron transfer from aldehydes by exploiting alcohol dehydrogenase 5 (ADH5). ADH5 is a highly conserved bifunctional enzyme serving as GSNO reductase (GSNOR) that down-regulates NO signaling and formaldehyde dehydrogenase (FDH) that detoxifies formaldehyde in the form of glutathione hemithioacetal. C174S mutation significantly reduced the supersulfidation of ADH5 and almost abolished GSNOR activity but spared FDH activity. Notably, Adh5C174S/C174S mice manifested improved cardiac functions possibly because of GSNOR elimination and consequent increased NO bioavailability. Therefore, we successfully separated dual functions (GSNOR and FDH) of ADH5 (mediated by the supersulfide catalysis) through the biochemical analysis for supersulfides in vitro and characterizing in vivo phenotypes of the GSNOR-deficient organisms that we established herein. Supersulfides in ADH5 thus constitute a substantial catalytic center for GSNO metabolism mediating electron transfer from aldehydes.

Persulfide Biosynthesis Conserved Evolutionarily in All Organisms.

Significance: Persulfides/polysulfides are sulfur-catenated molecular species (i.e., R-Sn-R', n > 2; R-Sn-H, n > 1, with R = cysteine, glutathione, and proteins), such as cysteine persulfide (CysSSH). These species are abundantly formed as endogenous metabolites in mammalian and human cells and tissues. However, the persulfide synthesis mechanism has yet to be thoroughly discussed. Recent Advances: We used ß-(4-hydroxyphenyl)ethyl iodoacetamide and mass spectrometry to develop sulfur metabolomics, a highly precise, quantitative analytical method for sulfur metabolites. Critical Issues: With this method, we detected appreciable amounts of different persulfide species in biological specimens from various organisms, from the domains Bacteria, Archaea, and Eukarya. By using our rigorously quantitative approach, we identified cysteinyl-tRNA synthetase (CARS) as a novel persulfide synthase, and we found that the CysSSH synthase activity of CARS is highly conserved from the domains Bacteria to Eukarya. Because persulfide synthesis is found not only with CARS but also with other sulfotransferase enzymes in many organisms, persulfides/polysulfides are expected to contribute as fundamental elements to substantially diverse biological phenomena. In fact, persulfide generation in higher organisms-that is, plants and animals-demonstrated various physiological functions that are mediated by redox signaling, such as regulation of energy metabolism, infection, inflammation, and cell death, including ferroptosis. Future Directions: Investigating CARS-dependent persulfide production may clarify various pathways of redox signaling in physiological and pathophysiological conditions and may thereby promote the development of preventive and therapeutic measures for oxidative stress as well as different inflammatory, metabolic, and neurodegenerative diseases. Antioxid. Redox Signal. 39, 983-999.

Supersulphides provide airway protection in viral and chronic lung diseases.

Supersulphides are inorganic and organic sulphides with sulphur catenation with diverse physiological functions. Their synthesis is mainly mediated by mitochondrial cysteinyl-tRNA synthetase (CARS2) that functions as a principal cysteine persulphide synthase (CPERS). Here, we identify protective functions of supersulphides in viral airway infections (influenza and COVID-19), in aged lungs and in chronic lung diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF). We develop a method for breath supersulphur-omics and demonstrate that levels of exhaled supersulphides increase in people with COVID-19 infection and in a hamster model of SARS-CoV-2 infection. Lung damage and subsequent lethality that result from oxidative stress and inflammation in mouse models of COPD, IPF, and ageing were mitigated by endogenous supersulphides production by CARS2/CPERS or exogenous administration of the supersulphide donor glutathione trisulphide. We revealed a protective role of supersulphides in airways with various viral or chronic insults and demonstrated the potential of targeting supersulphides in lung disease.

Presence of Helicobacter cinaedi in Atherosclerotic Abdominal Aortic Aneurysmal Wall.

Recently, the relationship between Helicobacter cinaedi (H. cinaedi) infection and several diseases, including cardiovascular and central nervous system disorders, bone and soft tissue disorders, and infectious abdominal aortic aneurysms (AAAs), has been reported. Moreover, H. cinaedi may be associated with arteriosclerosis. In the present study, we investigated the association between H. cinaedi infection and clinically uninfected AAAs. Genetic detection of H. cinaedi in the abdominal aneurysm wall was attempted in 39 patients with AAA undergoing elective open surgery between June 2019 and June 2020. DNA samples extracted from the arterial wall obtained during surgery were analyzed using nested polymerase chain reaction (PCR). The target gene region was the H. cinaedi-specific cytolethal distending toxin subunit B (cdtB). Nine (23.1%) of 39 patients showed positive bands corresponding to H. cinaedi, and further sequencing analyses demonstrated the presence of H. cinaedi DNAs in their aneurysm walls. In contrast, all the non-aneurysm arterial walls in our patients were negative for H. cinaedi. In conclusion, this is the first report of the detection of H. cinaedi in the walls of a clinically non-infectious AAA.

Synthesis of Sulfides and Persulfides Is Not Impeded by Disruption of Three Canonical Enzymes in Sulfur Metabolism.

Reactive sulfur species, or persulfides and polysulfides, such as cysteine hydropersulfide and glutathione persulfide, are endogenously produced in abundance in both prokaryotes and eukaryotes, including mammals. Various forms of reactive persulfides occur in both low-molecular-weight and protein-bound thiols. The chemical properties and great supply of these molecular species suggest a pivotal role for reactive persulfides/polysulfides in different cellular regulatory processes (e.g., energy metabolism and redox signaling). We demonstrated earlier that cysteinyl-tRNA synthetase (CARS) is a new cysteine persulfide synthase (CPERS) and is responsible for the in vivo production of most reactive persulfides (polysulfides). Some researchers continue to suggest that 3-mercaptopyruvate sulfurtransferase (3-MST), cystathionine ß-synthase (CBS), and cystathionine γ-lyase (CSE) may also produce hydrogen sulfide and persulfides that may be generated during the transfer of sulfur from 3-mercaptopyruvate to the cysteine residues of 3-MST or direct synthesis from cysteine by CBS/CSE, respectively. We thus used integrated sulfur metabolome analysis, which we recently developed, with 3-MST knockout (KO) mice and CBS/CSE/3-MST triple-KO mice, to elucidate the possible contribution of 3-MST, CBS, and CSE to the production of reactive persulfides in vivo. We therefore quantified various sulfide metabolites in organs derived from these mutant mice and their wild-type littermates via this sulfur metabolome, which clearly revealed no significant difference between mutant mice and wild-type mice in terms of reactive persulfide production. This result indicates that 3-MST, CBS, and CSE are not major sources of endogenous reactive persulfide production; rather, CARS/CPERS is the principal enzyme that is actually involved in and even primarily responsible for the biosynthesis of reactive persulfides and polysulfides in vivo in mammals.

Coupled discrete phase model and Eulerian wall film model for numerical simulation of respiratory droplet generation during coughing.

Computational fluid dynamics is widely used to simulate droplet-spreading behavior due to respiratory events. However, droplet generation inside the body, such as the number, mass, and particle size distribution, has not been quantitatively analyzed. The aim of this study was to identify quantitative characteristics of droplet generation during coughing. Airflow simulations were performed by coupling the discrete phase model and Eulerian wall film model to reproduce shear-induced stripping of airway mucosa. An ideal airway model with symmetric bifurcations was constructed, and the wall domain was covered by a mucous liquid film. The results of the transient airflow simulation indicated that the droplets had a wide particle size distribution of 0.1-400 µm, and smaller droplets were generated in larger numbers. In addition, the total mass and number of droplets generated increased with an increasing airflow. The total mass of the droplets also increased with an increasing mucous viscosity, and the largest number and size of droplets were obtained at a viscosity of 8 mPa s. The simulation methods used in this study can be used to quantify the particle size distribution and maximum particle diameter under various conditions.

Shining a light on SSP4: A comprehensive analysis and biological applications for the detection of sulfane sulfurs.

Fluorescent probes are useful tools for the detection of sulfane sulfurs in biological systems. In this work, we report the development of SSP4, a widely used probe generated in our laboratory. We describe its evolution, preparation, and physical/chemical properties. Fluorescence analyses of SSP4 determined its high selectivity and sensitivity to sulfane sulfurs, even with the interfering presence of other species, such as amino acids and metal ions. Protocols for using SSP4 in a relatively quick and simple manner for the detection of persulfidated proteins, including papain, BSA, and GAPDH were developed. The method was then applied to human protein disulfide isomerase (PDI), leading to the discovery that persulfidation can occur at PDI's non-active site cysteines, and that PDI reductase activity is affected by sulfane sulfur treatment. Protocols for using SSP4 for the bioimaging of exogenous and endogenous sulfane sulfurs in different -cell lines were also established. These results should guide further applications of SSP4.

Chemical Biology of Reactive Sulfur Species: Hydrolysis-Driven Equilibrium of Polysulfides as a Determinant of Physiological Functions.

Significance: Polysulfide species (i.e., R-Sn-R', n > 2; and R-Sn-H, n > 1) exist in many organisms. The highly nucleophilic nature of hydropersulfides and hydropolysulfides contributes to the potent antioxidant activities of polysulfide species that protect organisms against oxidative and electrophilic stresses. Recent Advances: Accumulating evidence suggests that organic polysulfides (R-Sn-R') readily undergo alkaline hydrolysis, which results in formation of both nucleophilic hydrosulfide/polysulfide (R-Sn-1H) and electrophilic sulfenic acid (R'SOH) species. Polysulfides maintain a steady-state equilibrium that is driven by hydrolysis even in aqueous physiological milieus. This unique property makes polysulfide chemistry and biology more complex than previously believed. Critical Issues: The hydrolysis equilibrium of polysulfides shifts to the right when electrophiles are present. Strong electrophilic alkylating agents (e.g., monobromobimane) greatly enhance polysulfide hydrolysis, which leads to increased polysulfide degradation and artifactual formation of bis-S-bimane adducts in the absence of free hydrogen sulfide. The finding that hydroxyl group-containing substances such as tyrosine efficiently protected polysulfides from hydrolysis led to development of the new alkylating agent, N-iodoacetyl l-tyrosine methyl ester (TME-IAM). TME-IAM efficiently and specifically traps and stabilizes hydropolysulfides and protects polysulfide chains from hydrolysis, and, when used with mass spectrometry, TME-IAM allows speciation of the reactive sulfur metabolome. In addition, the polyethylene glycol-conjugated maleimide-labeling gel shift assay, which relies on unique hydrolysis equilibrium of polysulfides, will be a reliable technique for proteomics of polysulfide-containing proteins. Future Directions: Using precise methodologies to achieve a better understanding of the occurrence and metabolism of polysulfide species is necessary to gain insights into the undefined biology of polysulfide species. Antioxid. Redox Signal. 36, 327-336.

GRIM-19 is a target of mycobacterial Zn2+ metalloprotease 1 and indispensable for NLRP3 inflammasome activation.

Tuberculosis is a communicable disease caused by Mycobacterium tuberculosis which primarily infects macrophages and establishes intracellular parasitism. A mycobacterial virulence factor Zn2+ metalloprotease 1 (Zmp1) is known to suppress interleukin (IL)-1ß production by inhibiting caspase-1 resulting in phagosome maturation arrest. However, the molecular mechanism of caspase-1 inhibition by Zmp1 is still elusive. Here, we identified GRIM-19 (also known as NDUFA13), an essential subunit of mitochondrial respiratory chain complex I, as a novel Zmp1-binding protein. Using the CRISPR/Cas9 system, we generated GRIM-19 knockout murine macrophage cell line J774.1 and found that GRIM-19 is essential for IL-1ß production during mycobacterial infection as well as in response to NLRP3 inflammasome-activating stimuli such as extracellular ATP or nigericin. We also found that GRIM-19 is required for the generation of mitochondrial reactive oxygen species and NLRP3-dependent activation of caspase-1. Loss of GRIM-19 or forced expression of Zmp1 resulted in a decrease in mitochondrial membrane potential. Our study revealed a previously unrecognized role of GRIM-19 as an essential regulator of NLRP3 inflammasome and a molecular mechanism underlying Zmp1-mediated suppression of IL-1ß production during mycobacterial infection.

Methods in sulfide and persulfide research.

Sulfides and persulfides/polysulfides (R-Sn-R', n > 2; R-Sn-H, n > 1) are endogenously produced metabolites that are abundant in mammalian and human cells and tissues. The most typical persulfides that are widely distributed among different organisms include various reactive persulfides-low-molecular-weight thiol compounds such as cysteine hydropersulfide, glutathione hydropersulfide, and glutathione trisulfide as well as protein-bound thiols. These species are generally more redox-active than are other simple thiols and disulfides. Although hydrogen sulfide (H2S) has been suggested for years to be a small signaling molecule, it is intimately linked biochemically to persulfides and may actually be more relevant as a marker of functionally active persulfides. Reactive persulfides can act as powerful antioxidants and redox signaling species and are involved in energy metabolism. Recent evidence revealed that cysteinyl-tRNA synthetases (CARSs) act as the principal cysteine persulfide synthases in mammals and contribute significantly to endogenous persulfide/polysulfide production, in addition to being associated with a battery of enzymes including cystathionine ß-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, which have been described as H2S-producing enzymes. The reactive sulfur metabolites including persulfides/polysulfides derived from CARS2, a mitochondrial isoform of CARS, also mediate not only mitochondrial biogenesis and bioenergetics but also anti-inflammatory and immunomodulatory functions. The physiological roles of persulfides, their biosynthetic pathways, and their pathophysiology in various diseases are not fully understood, however. Developing basic and high precision techniques and methods for the detection, characterization, and quantitation of sulfides and persulfides is therefore of great importance so as to thoroughly understand and clarify the exact functions and roles of these species in cells and in vivo.

Sulfide catabolism ameliorates hypoxic brain injury.

The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain's sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury.

Induction of Fos expression in the rat brain after intragastric administration of dried bonito dashi.

Objectives: Dried bonito dashi, a traditional Japanese fish broth made from dried bonito tuna, enhances food palatability due to its specific umami flavor characteristics. However, the pattern of brain activation following dashi ingestion has not been previously investigated.Methods: We mapped activation sites of the rat brain after intragastric loads of dried bonito dashi by measuring neuronal levels of the Fos protein, a functional marker of neuronal activation.Results: Compared to intragastric saline, intragastric dashi administration produced enhanced Fos expression in four forebrain regions: the medial preoptic area, subfornical organ, habenular nucleus, and central nucleus of the amygdala. Interestingly, the medial preoptic area was found to be the only feeding-related hypothalamic area responsive to dashi administration. Moreover, dashi had no effect in the nucleus accumbens and ventral tegmental area, two connected sites known to be activated by highly palatable sugars and fats. In the hindbrain, dashi administration produced enhanced Fos expression in both visceral sensory (caudal nucleus of the solitary tract, dorsal part of the lateral parabrachial nucleus, and area postrema) and autonomic (rostral ventrolateral medulla, and caudal ventrolateral medulla) sites.Discussion: The results demonstrate the activation of discrete forebrain and hindbrain regions following intragastric loads of dried bonito dashi. Our data suggest that the gut-brain axis is the principal mediator of the postingestive effects associated with the ingestion of dashi.

Associations between dietary consumption and sleep quality in young Japanese males.

PURPOSE: Poor sleep quality has been reported to be a risk factor for cardiovascular disease, diabetes, and metabolic syndrome, as well as mental disorders including depression and anxiety. However, few studies have investigated the association between sleep quality and diet in young males. We aimed to assess this association, adjusting for psychological factors. METHODS: In this study, a total of 124 male Japanese students were analyzed. Sleep quality, diet, and psychological symptoms were assessed using self-reported questionnaires, including the Pittsburgh Sleep Quality Index (PSQI), brief-type self-administered diet history questionnaire (BDHQ), 12-item General Health Questionnaire (GHQ12), and State-Trait Anxiety Inventory (STAI) A-Trait scale. RESULTS: Among participants, 40% exhibited a PSQI total score ≥ 6, indicating poor sleep quality. Poor sleep quality was associated with poor mental health status and higher levels of anxiety. After adjusting for covariates including these psychological factors, poor sleep quality was significantly associated with low intakes of fat, beta-carotene, retinol, alpha-tocopherol, vitamin K, vitamin B1, daidzein, genistein, and iron. Poor sleep quality was also associated with low intake of pulses, fat and oil, as well as high intakes of sugar-sweetened beverages. CONCLUSIONS: Our findings demonstrated that sleep quality among young Japanese males was associated with specific dietary features, independently of psychological status, which may help to elucidate the mechanisms underlying the link between sleep and sleep-related diseases.

Intake and preference for dried bonito dashi in male Sprague-Dawley rats and C57BL/6 N mice.

Dried bonito dashi is a traditional Japanese fish broth that enhances palatability of various dishes due to its specific flavor. The present study examined influences of dietary fat levels (10% vs. 45% fat), presentation order of dried bonito dashi (ascending vs. descending concentrations), and prior experience with dashi on subsequent dashi intake and preference using two-bottle choice tests in two rodent strains, Sprague-Dawley (SD) rats and C57BL/6 N (B6N) mice. In the ascending concentration tests, SD rats on a low fat diet preferred 10-100% dashi to water, whereas B6N mice showed a blunted preference for dashi. Consumption of a high fat diet reduced dashi preference in SD rats. The B6N mice on the high fat diet never preferred dashi at any concentration. In the descending concentration tests, SD rats on the low fat diet preferred dashi over a wide range (0.03-100% dashi). The B6N mice showed a trend similar to that of SD rats. Ingestion of the high fat diet in both strains reduced dashi preference in the descending concentration tests. However, introduction of the high fat diet to dashi experienced rats maintained on the low fat diet, reduced neither dashi intake nor dashi preference. Dashi intake affected neither high fat diet intake, caloric intake, nor preference for high fat diet. These results suggest that preference for dried bonito dashi is influenced at least by 1) dietary fat levels, 2) presentation order of dashi, and 3) prior experience with dashi.

Rational Design of a Dual-Reactivity-Based Fluorescent Probe for Visualizing Intracellular HSNO.

Thionitrous acid (HSNO), the smallest S-nitrosothiol, is emerging as a potential key intermediate in cellular redox regulation linking two signaling molecules H2 S and NO. However, the chemical biology of HSNO remains poorly understood. A major hurdle is the lack of methods for selective detection of HSNO in biological systems. Herein, we report the rational design, synthesis, and evaluation of the first fluorescent probe TAP-1 for HSNO detection. TAP-1 showed high selectivity and sensitivity to HSNO in aqueous media and cells, providing a useful tool for understanding the functions of HSNO in biology.

Mitochondrial cysteinyl-tRNA synthetase is expressed via alternative transcriptional initiation regulated by energy metabolism in yeast cells.

Eukaryotes typically utilize two distinct aminoacyl-tRNA synthetase isoforms, one for cytosolic and one for mitochondrial protein synthesis. However, the genome of budding yeast (Saccharomyces cerevisiae) contains only one cysteinyl-tRNA synthetase gene (YNL247W, also known as CRS1). In this study, we report that CRS1 encodes both cytosolic and mitochondrial isoforms. The 5' complementary DNA end method and GFP reporter gene analyses indicated that yeast CRS1 expression yields two classes of mRNAs through alternative transcription starts: a long mRNA containing a mitochondrial targeting sequence and a short mRNA lacking this targeting sequence. We found that the mitochondrial Crs1 is the product of translation from the first initiation AUG codon on the long mRNA, whereas the cytosolic Crs1 is produced from the second in-frame AUG codon on the short mRNA. Genetic analysis and a ChIP assay revealed that the transcription factor heme activator protein (Hap) complex, which is involved in mitochondrial biogenesis, determines the transcription start sites of the CRS1 gene. We also noted that Hap complex-dependent initiation is regulated according to the needs of mitochondrial energy production. The results of our study indicate energy-dependent initiation of alternative transcription of CRS1 that results in production of two Crs1 isoforms, a finding that suggests Crs1's potential involvement in mitochondrial energy metabolism in yeast.