Eukaryotic Mitochondria Research Articles

Insights into Fungal Mitochondrial Genomes and Inheritance Based on Current Findings from Yeast-like Fungi.

The primary functions of mitochondria are to produce energy and participate in the apoptosis of cells, with them being highly conserved among eukaryotes. However, the composition of mitochondrial genomes, mitochondrial DNA (mtDNA) replication, and mitochondrial inheritance varies significantly among animals, plants, and fungi. Especially in fungi, there exists a rich diversity of mitochondrial genomes, as well as various replication and inheritance mechanisms. Therefore, a comprehensive understanding of fungal mitochondria is crucial for unraveling the evolutionary history of mitochondria in eukaryotes. In this review, we have organized existing reports to systematically describe and summarize the composition of yeast-like fungal mitochondrial genomes from three perspectives: mitochondrial genome structure, encoded genes, and mobile elements. We have also provided a systematic overview of the mechanisms in mtDNA replication and mitochondrial inheritance during bisexual mating. Additionally, we have discussed and proposed open questions that require further investigation for clarification.

Lactate oxidation in Paracoccus denitrificans

Paracoccus denitrificans has a classical cytochrome-dependent electron transport chain and two alternative oxidases. The classical transport chain is very similar to that in eukaryotic mitochondria. Thus, P. denitrificans can serve as a model of the mammalian mitochondrion that may be more tractable in elucidating mechanisms of regulation of energy production than are mitochondria. In a previous publication we reported detailed studies on respiration in P. denitrificans grown aerobically on glucose or malate. We noted that P. denitrificans has large stores of lactate under various growth conditions. This is surprising because P. denitrificans lacks an NAD+-dependent lactate dehydrogenase. The aim of this study was to investigate the mechanisms of lactate oxidation in P. denitrificans. We found that the bacterium grows well on either d-lactate or l-lactate. Growth on lactate supported a rate of maximum respiration that was equal to that of cells grown on glucose or malate. We report proteomic, metabolomic, and biochemical studies that establish that the metabolism of lactate by P. denitrificans is mediated by two non-NAD+-dependent lactate dehydrogenases. One prefers d-lactate over l-lactate (D-iLDH) and the other prefers l-lactate (L-iLDH). We cloned and produced the D-iLDH and characterized it. The Km for d-lactate was 34 μM, and for l-lactate it was 3.7 mM. Pyruvate was not a substrate, rendering the reaction unidirectional with lactate being converted to pyruvate for entry into the TCA cycle. The intracellular lactate was ∼14 mM such that both isomers could be metabolized by the enzyme. The enzyme has 1 FAD per molecule and utilizes a quinone rather than NAD + as an electron acceptor. D-iLDH provides a direct entry of lactate reducing equivalents into the cytochrome chain, potentially explaining the high respiratory capacity of P. denitrificans in the presence of lactate.

Bacterial Electrophysiology.

Bacterial ion fluxes are involved in the generation of energy, transport, and motility. As such, bacterial electrophysiology is fundamentally important for the bacterial life cycle, but it is often neglected and consequently, by and large, not understood. Arguably, the two main reasons for this are the complexity of measuring relevant variables in small cells with a cell envelope that contains the cell wall and the fact that, in a unicellular organism, relevant variables become intertwined in a nontrivial manner. To help give bacterial electrophysiology studies a firm footing, in this review, we go back to basics. We look first at the biophysics of bacterial membrane potential, and then at the approaches and models developed mostly for the study of neurons and eukaryotic mitochondria. We discuss their applicability to bacterial cells. Finally, we connect bacterial membrane potential with other relevant (electro)physiological variables and summarize methods that can be used to both measure and influence bacterial electrophysiology.

Testing a Hypothesis of 12S rRNA Methylation by Putative METTL17 Methyltransferase.

Mitochondrial ribosome assembly is a complex multi-step process involving many additional factors. Ribosome formation differs in various groups of organisms. However, there are universal steps of assembly and conservative factors that have been retained in evolutionarily distant taxa. METTL17, the object of the current study, is one of these conservative factors involved in mitochondrial ribosome assembly. It is present in both bacteria and the mitochondria of eukaryotes, in particular mice and humans. In this study, we tested a hypothesis of putative METTL17 methyltransferase activity. MALDI-TOF mass spectrometry was used to evaluate the methylation of a putative METTL17 target - a 12S rRNA region interacting with METTL17 during mitochondrial ribosome assembly. The investigation of METTL17 and other mitochondrial ribosome assembly factors is of both fundamental and practical significance, because defects in mitochondrial ribosome assembly are often associated with human mitochondrial diseases.

Structural bioinformatics studies of bacterial outer membrane beta-barrel transporters and their AlphaFold2 predicted water-soluble QTY variants.

Beta-barrel outer membrane proteins (OMP) are integral components of Gram-negative bacteria, eukaryotic mitochondria, and chloroplasts. They play essential roles in various cellular processes including nutrient transport, membrane stability, host-pathogen interactions, antibiotic resistance and more. The advent of AlphaFold2 for accurate protein structure predictions transformed structural bioinformatic studies. We previously used a QTY code to convert hydrophobic alpha-helices to hydrophilic alpha-helices in over 50 membrane proteins with all alpha-helices. The QTY code systematically replaces hydrophobic leucine (L), isoleucine (I), valine (V), and phenylalanine (F) with hydrophilic glutamine (Q), threonine (T), and tyrosine (Y). We here present a structural bioinformatic analysis of five outer membrane beta-barrel proteins with known molecular structures, including a) BamA, b) Omp85 (also called Sam50), c) FecA, d) Tsx, and e) OmpC. We superposed the structures of five native beta-barrel outer membrane proteins and their AlphaFold2-predicted corresponding QTY variant structures. The superposed structures of OMPs and their QTY variants exhibit remarkable structural similarity, as evidenced by residue mean square distance (RMSD) values between 0.206Å to 0.414Å despite the replacement of at least 22% (Transmembrane variation) of the amino acids in the transmembrane regions. We also show that native outer membrane proteins and QTY variants have different hydrophobicity patches. Our study provides important insights into the differences between hydrophobic and hydrophilic beta-barrels and validates the QTY code for studying beta-barrel membrane proteins and perhaps other hydrophobic aggregated proteins. Our findings demonstrate that the QTY code can be used as a simple tool for designing hydrophobic proteins in various biological contexts.

Copper exerts cytotoxicity through inhibition of iron-sulfur cluster biogenesis on ISCA1/ISCA2/ISCU assembly proteins.

Copper is an essential mineral nutrient that provides the cofactors for some key enzymes. However, excess copper is paradoxically cytotoxic. Wilson's disease is an autosomal recessive hereditary disease characterized by pathological copper accumulation in many organs, with high mortality and disability. Nevertheless, many questions about the molecular mechanism in Wilson's disease remain unknown and there is an imperative need to address these questions to better exploit therapeutic strategy. In this study, we constructed the mouse model of Wilson's disease, ATP7A-/- immortalized lymphocyte cell line and ATP7B knockdown cells to explore whether copper could impair iron-sulfur cluster biogenesis in eukaryotic mitochondria. Through a series of cellular, molecular, and pharmacological analyses, we demonstrated that copper could suppress the assembly of Fe-S cluster, decrease the activity of the Fe-S enzyme and disorder the mitochondrial function both in vivo and in vitro. Mechanistically, we found that human ISCA1, ISCA2 and ISCU proteins have a strong copper-binding activity, which would hinder the process of iron-sulfur assembly. Of note, we proposed a novel mechanism of action to explain the toxicity of copper by providing evidence that iron-sulfur cluster biogenesis may be a primary target of copper toxicity both in cells and mouse models. In summary, the current work provides an in-depth study on the mechanism of copper intoxication and describes a framework for the further understanding of impaired Fe-S assembly in the pathological processes of Wilson's diseases, which helps to develop latent therapeutic strategies for the management of copper toxicity.

Multiomics analyses reveals Anaplasma phagocytophilum Ats-1 induces anti-apoptosis and energy metabolism by upregulating the respiratory chain-mPTP axis in eukaryotic mitochondria

BackgroundAnaplasma translocated substrate 1 (Ats-1) is an effector of type 4 secretory systems (T4SS) and the main virulence factor of Anaplasma phagocytophilum. Ats-1 is involved in the regulation of host cell biological processes, but the specific molecular mechanism of its action is unclear.ResultsIn this study, we identified Ats-1 as involved in mitochondrial respiratory regulation of HEK293T cells by multi-omics analysis. After intracellular expression of Ats-1, adenosine triphosphate levels and the proliferation of HEK293T cells were both up-regulated, while HEK293T cells apoptosis was inhibited. Ats-1 targeted translocation to the mitochondria where it up-regulated the expression of NDUFB5, NDUFB3, NDUFS7, COX6C, and SLC25A5, thereby enhancing energy production and inhibiting HEK293T cells apoptosis while enhancing HEK293T cells proliferation, and ultimately facilitating Anaplasma phagocytophilum replication in HEK293T cells.ConclusionsThis study demonstrated that Anaplasma phagocytophilum Ats-1 induces anti-apoptosis and energy metabolism by upregulating the respiratory chain-mPTP axis in eukaryotic mitochondria. These results provide a better understanding of the pathogenic mechanism of Anaplasma phagocytophilum within host cells.

Ion selectivity and gating behavior of the CorA-type channel Bpss1228.

Magnesium is an essential element to sustain all forms of life. Total intracellular magnesium content is determined by the balance of magnesium influx and efflux. CorA is a divalent selective channel in the metal ion transport superfamily and is the major Mg2+ uptake pathway in prokaryotes and eukaryotic mitochondria. Previous studies have demonstrated that CorA showed distinct magnesium bound closed conformation and Mg2+-free states. In addition, CorA is regulated by cytoplasmic magnesium ions and its gating mechanism has been investigated by electron paramagnetic resonance technique and molecular dynamic simulations. Here, we report a study of the putative CorA-type channel Bpss1228 from Burkholderia pseudomallei, which has been shown to be significantly associated with pseudomallei infection. We expressed and purified the Bpss1228 in full-length. Subsequently, electrophysiological experiments further investigated the electrical characteristics of Bpss1228 and revealed that it was a strictly cation-selective channel. We also proved that Bpss1228 not only possessed magnesium-mediated regulatory property a remarkable ability to be modulated by magnesium ions. Finally, we observed the three-step gating behavior of Bpss1228 on planar lipid bilayer, and further proposed a synergistic gating mechanism by which CorA family channels control intracellular magnesium homeostasis.

Ancestral State Reconstructions Trace Mitochondria But Not Phagocytosis to the Last Eukaryotic Common Ancestor.

Two main theories have been put forward to explain the origin of mitochondria in eukaryotes: phagotrophic engulfment (undigested food) and microbial symbiosis (physiological interactions). The two theories generate mutually exclusive predictions about the order in which mitochondria and phagocytosis arose. To discriminate the alternatives, we have employed ancestral state reconstructions (ASR) for phagocytosis as a trait, phagotrophy as a feeding habit, the presence of mitochondria, the presence of plastids, and the multinucleated organization across major eukaryotic lineages. To mitigate the bias introduced by assuming a particular eukaryotic phylogeny, we reconstructed the appearance of these traits across 1789 different rooted gene trees, each having species from opisthokonts, mycetozoa, hacrobia, excavate, archeplastida, and Stramenopiles, Alveolates and Rhizaria. The trees reflect conflicting relationships and different positions of the root. We employed a novel phylogenomic test that summarizes ASR across trees which reconstructs a last eukaryotic common ancestor that possessed mitochondria, was multinucleated, lacked plastids, and was non-phagotrophic as well as non-phagocytic. This indicates that both phagocytosis and phagotrophy arose subsequent to the origin of mitochondria, consistent with findings from comparative physiology. Furthermore, our ASRs uncovered multiple origins of phagocytosis and of phagotrophy across eukaryotes, indicating that, like wings in animals, these traits are useful but neither ancestral nor homologous across groups. The data indicate that mitochondria preceded the origin of phagocytosis, such that phagocytosis cannot have been the mechanism by which mitochondria were acquired.

Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation.

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.

Molecular Mechanism of Citrate Efflux by the Mitochondrial Citrate Transporter CT in Filamentous Fungus Mucor circinelloides WJ11.

The mitochondrial citrate transporter (MCT) plays an important role in citrate efflux from the mitochondria in eukaryotes, and hence provides a direct correlation between carbohydrate metabolism and lipid synthesis. Our previous studies on transporters confirmed the presence of two MCTs (TCT and CT) in oleaginous Mucor circinelloides WJ11 associated with high lipid accumulation. However, the molecular mechanism of citrate efflux from the mitochondria by MCT in M. circinelloides is still unclear. To study the citrate transport mechanism of CT, the citrate transporter gene was expressed in Escherichia coli, and its product was purified. The citrate transport activity of the protein was studied in CT reconstituted liposomes. Our results showed high efficiency of CT for [14C] citrate/citrate exchange with K m 0.01 mM at 25°C. Besides citrate, other molecules such as oxaloacetate, malate, fumarate, succinate aconitate, oxoadipate, isocitrate, and glutamate also promote citrate transport. In addition, the ct overexpression and knockout plasmids were constructed and transferred into M. circinelloides WJ11, and the mitochondria were isolated, and the transport activity was studied. Our findings showed that in the presence of 10 mM malate, the mitochondria of ct-overexpressing transformant showed 51% increase in the efflux rate of [14C] citrate, whereas the mitochondria of the ct-knockout transformant showed 18% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. This study provided the first mechanistic evidence of citrate efflux from the mitochondria by citrate transporter in oleaginous filamentous fungus M. circinelloides, which is associated with high lipid accumulation.

Asymmetric CorA Gating Mechanism as Observed by Molecular Dynamics Simulations.

The CorA family of proteins plays a housekeeping role in the homeostasis of divalent metal ions in many bacteria and archaea as well as in mitochondria of eukaryotes, rendering it an important target to study the mechanisms of divalent transport and regulation across different life domains. Despite numerous studies, the mechanistic details of the channel gating and the transport of the metal ions are still not entirely understood. Here, we use all-atom and coarse-grained molecular dynamics simulations combined with in vitro experiments to investigate the influence of divalent cations on the function of CorA. Simulations reveal pronounced asymmetric movements of monomers that enable the rotation of the α7 helix and the cytoplasmic subdomain with the subsequent formation of new interactions and the opening of the channel. These computational results are functionally validated using site-directed mutagenesis of the intracellular cytoplasmic domain residues and biochemical assays. The obtained results infer a complex network of interactions altering the structure of CorA to allow gating. Furthermore, we attempt to reconcile the existing gating hypotheses for CorA to conclude the mechanism of transport of divalent cations via these proteins.

Microbiome-Mitochondria Communication in the Regulation of Host Longevity

Mitochondria are ancient relatives of bacteria in eukaryotic cells and dynamically interconnected through organelle fusion and fission. Given the close relationship between bacteria and eukaryotic mitochondria during evolution, my group is interested in understanding the critical role of their communication in regulating host’s longevity. We have conducted genome-scale screens to decipher how bacterial genetic composition impacts host longevity, leading to the discovery of specific bacteria-secreted metabolites that fine-tune the mitochondrial fusion-fission balance and consequently promotes longevity across different host species. We have further developed optogenetic approaches to manipulate bacterial gene expression and metabolite production inside the gut of live organisms, in order to investigate the microbiome-mitochondria communication in time and space. Our studies demonstrate a novel mode of signaling communication between bacteria and mitochondria, reveal its vital impacts on host healthy aging, and provide new methods to decipher the spatiotemporal relationship between the microbiome and the host.

Output Regulation and Function Optimization of Mitochondria in Eukaryotes.

The emergence of endosymbiosis between aerobic alpha-proteobacterium and anaerobic eukaryotic cell precursors opened the chapter of eukaryotic evolution. Multiple functions of mitochondria originated from the ancient precursors of mitochondria and underwent remodeling in eukaryotic cells. Due to the dependence on mitochondrial functions, eukaryotic cells need to constantly adjust mitochondrial output based on energy demand and cellular stress. Meanwhile, eukaryotes conduct the metabolic cooperation between different cells through the involvement of mitochondria. Under some conditions, mitochondria might also be transferred to nearby cells to provide a protective mechanism. However, the endosymbiont relationship determines the existence of various types of mitochondrial injury, such as proteotoxic stress, mutational meltdown, oxidative injure, and immune activation caused by released mitochondrial contents. Eukaryotes have a repertoire of mitochondrial optimization processes, including various mitochondrial quality-control proteins, regulation of mitochondrial dynamics and activation of mitochondrial autophagy. When these quality-control processes fail, eukaryotic cells can activate apoptosis to intercept uncontrolled cell death, thereby minimizing the damage to extracellular tissue. In this review, we describe the intracellular and extracellular context-based regulation of mitochondrial output in eukaryotic cells, and introduce new findings on multifaceted quality-control processes to deal with mitochondrial defects.

The Origin of Mitochondria and their Role in the Evolution of Life and Human Health

In studies of many medical and biological problems, there is a clear underestimation of the fundamental role of mitochondria in the evolution of eukaryotic organisms on the planet, including fungi, plants and fauna. It is important to take into consideration numerous fundamental functions of mitochondria when studying the human physiology and pathology, aging mechanisms. In this lecture, we briefly discuss the origin of mitochondria and their importance in the emergence of plants, which appeared on the planet 1–1.5 billion years later than eukaryotes with mitochondria, and served as the food basis for the rapid evolution of all species of the animal world. In the course of transformation of protobacteria into mitochondria, approximately 1000–1500 genes were transferred to the nucleus of eukaryotes, and the remaining 37 mitochondrial genes (mtDNA) exist in all types of animals. The presence of mitochondria in eukaryotes led to increased production of reactive oxygen species and accelerated mutations in mtDNA. The spread of sexual reproduction may have been the way of protection against the accumulation of harmful mutations in mtDNA. At the same time, in all animal species mtDNA is inherited only maternally. In humans, the maternal inheritance of mtDNA led to uneven distribution, in small or inbred populations in particular, of a number of diseases with large prevalence in men, as well as male infertility, and in general accelerated aging and shorter lifespan in men, as compared with women. These negative consequences of the maternal inheritance of mtDNA were termed as “mother’s curse”. Recent studies have shown that mtDNA mutations are not the cause of human aging. The crisis of the mitochondrial free radical theory of aging is largely associated with the neglect of the protonated form of the superoxide radical, the perhydroxyl radical, which activates the peroxidation of polyunsaturated fatty acids in mitochondrial phospholipids. This results in production of various biologically active molecules and toxins. Therefore, initially aging may lack the manifestation of specific symptoms because of gradual accumulation of a wide variety of disorders and damages to the structure and functions of mitochondria and cells that finally lead to the development of diseases. The pathologies appear primarily in those organs that have a wide range of functional activity and a high dependence on oxygen consumption, namely the heart, brain, skeletal muscles and vascular epithelium.

Evolution of the human mitochondrial ABCB7 [2Fe–2S](GS)4 cluster exporter and the molecular mechanism of an E433K disease-causing mutation

Iron-sulfur cluster proteins play key roles in a multitude of cellular processes. Iron-sulfur cofactors are assembled primarily in mitochondria and are then exported to the cytosol by use of an ABCB7 transporter. It has been shown that the yeast mitochondrial transporter Atm1 can export glutathione-coordinated iron-sulfur clusters, [2Fe–2S](SG)4, providing a source of cluster units for cytosolic iron-sulfur cluster assembly systems. This pathway is consistent with the endosymbiotic model of mitochondrial evolution where homologous bacterial heavy metal transporters, utilizing metal glutathione adducts, were adapted for use in eukaryotic mitochondria. Herein, the basis for endosymbiotic evolution of the human cluster export protein (ABCB7) is developed through a BLAST analysis of transporters from ancient proteobacteria. In addition, a functional comparison of native human protein, versus a disease-causing mutant, demonstrates a key role for residue E433 in promoting cluster transport. Dysfunction in mitochondrial export of Fe–S clusters is a likely cause of the disease condition X-linked sideroblastic anemia.

Mitochondrial Calcium Uniporter (MCU) deficiency reveals an alternate path for Ca2+ uptake in photoreceptor mitochondria

Rods and cones use intracellular Ca2+ to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca2+ entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca2+ uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu-/- zebrafish and a rod photoreceptor-specific Mcu-/- mouse. Using genetically encoded Ca2+ sensors to directly examine Ca2+ uptake in zebrafish cone mitochondria, we found that loss of MCU reduces but does not eliminate mitochondrial Ca2+ uptake. Loss of MCU does not lead to photoreceptor degeneration, mildly affects mitochondrial metabolism, and does not alter physiological responses to light, even in the absence of the Na+/Ca2+, K+ exchanger. Our results reveal that MCU is dispensable for vertebrate photoreceptor function, consistent with its low expression and the presence of an alternative pathway for Ca2+ uptake into photoreceptor mitochondria.

What is the Role of Lipid Membrane-embedded Quinones in Mitochondria and Chloroplasts? Chemiosmotic Q-cycle versus Murburn Reaction Perspective.

Quinones are found in the lipid membranes of prokaryotes like E. coli and cyanobacteria, and are also abundant in eukaryotic mitochondria and chloroplasts. They are intricately involved in the reaction mechanism of redox phosphorylations. In the Mitchellian chemiosmotic school of thought, membrane-lodged quinones are perceived as highly mobile conveyors of two-electron equivalents from the first leg of Electron Transport Chain (ETC) to the 'second pit-stop' of Cytochrome bc1 or b6f complex (CBC), where they undergo a regenerative 'Q-cycle'. In Manoj's murburn mechanism, the membrane-lodged quinones are perceived as relatively slow-moving one- or two- electron donors/acceptors, enabling charge separation and the CBC resets a one-electron paradigm via 'turbo logic'. Herein, we compare various purviews of the two mechanistic schools with respect to: constraints in mobility, protons' availability, binding of quinones with proteins, structural features of the protein complexes, energetics of reaction, overall reaction logic, etc. From various perspectives, the murburn mechanism appeals as a viable alternative explanation well-rooted in thermodynamics/kinetics and one which lends adequate structure-function correlations for the roles of quinones, lipid membrane and associated proteins.

A Review on the Possible Leakage of Electrons through the Electron Transport Chain within Mitochondria

The finding of electron leakage during the electron transport within the mitochondrial membrane (in eukaryotes) or in the cell membrane of the prokaryotes is an important issue for the accumulation of the Reactive Oxygen Species (ROS) in the cytosol which in turn induce the probable aging of cells. In eukaryotes, mitochondrion is known to be the major site of the ROS generation in different pathological processes which may further cause cell damages as evident through the ischemia-reperfusion (I/R) injury, respiratory diseases, cell apoptosis, and even the onset of cancer. Thus, the mitochondrial leakage and the physiological effect of leaked protons and electrons grow up with future interest in energy metabolism. Current review focused on the physiological impact of electron/ proton leakage particularly in the eukaryotic cells based on the previous reports; emphasized on the prospects of the eukaryotic mitochondrion as a modulator of proton and electron leakage; and finally attempted to assess the regulatory mechanisms of such electron/ proton leakage.

Genome-wide effect of tetracycline, doxycycline and 4-epidoxycycline on gene expression in Saccharomyces cerevisiae.

Tetracycline (Tet) and derivative chemicals (e.g., doxycycline or Dox) have gained widespread recognition for their antibiotic properties since their introduction in the late 1970s, but recent work with these chemicals in the lab has shifted to include multiple techniques in all genetic model systems for the precise control of gene expression. The most widely used Tet‐modulated methodology is the Tet‐On/Tet‐Off gene expression system. Tet is generally considered to have effects specific to bacteria; therefore, it should have few off‐target effects when used in eukaryotic systems, and a previous study in the yeast Saccharomyces cerevisiae found that Dox had no effect on genome‐wide gene expression as measured by microarray. In contrast, another study found that the use of Dox in common cell lines and several model organisms led to mitonuclear protein imbalance, suggesting an inhibitory role of Dox in eukaryotic mitochondria. Recently, a new Dox derivative, 4‐epidoxycycline (4‐ED) was developed that was shown to have less off‐target consequences on mitochondrial health. To determine the best tetracycline family chemical to use for gene expression control in S. cerevisiae, we performed RNA sequencing (RNA‐seq) on yeast grown on standard medium compared with growth on media supplemented with Tet, Dox or 4‐ED. We found each caused dozens of genes to change expression, with Dox eliciting the greatest expression responses, suggesting that the specific tetracycline used in experiments should be tailored to the specific gene(s) of interest when using the Tet‐On/Tet‐Off system to reduce the consequences of confounding off‐target responses.