Phenotypes In Yeast Research Articles

Identification new potential multidrug resistance proteins of Saccharomyces cerevisiae

ABC (ATP-binding cassette) proteins can transport metabolic molecules and removes metabolic products and xenobiotics from the cell. The important problem is to study activity and search inhibitors of ABC proteins. There is a problem that ABC-proteins can transport hydrophobic drugs across the cell membrane due to their high substrate specificity. According to published data, Saccharomyces cerevisiae is an ideal model organism for analysis a lot of functional processes and gene activities of human cells. The aim of the present work is to reveal new potential yeast MDR proteins in S. cerevisiae with novel approach based on the cluster analysis.According to the cluster analysis of yeast ABCB subfamily, STE6 protein is turned out to be the most related to human P-gp protein. The largest number of homologues with human MDR proteins was found in the yeast ABCC subfamily. Yeast BPT1 and YCF1 proteins are shown to be the most phylogenetically close to human MRP1. In the ABCG subfamily of yeast, PDR10, PDR12, PDR15 and PDR18 are turned out to be potential proteins of multidrug resistance. The future experimental study of these subfamilies should be conducted in order to confirm the role of STE6, YCF1, BPT1, PDR10, PDR12, PDR15 and PDR18 in MDR phenotype of yeast and to study their activity modulators.

Article of Significant Interest in This Issue

Maintenance of protein homeostasis requires the functions of Hsp70 chaperones. Kao et al. (e00628-19) investigate an evolutionarily conserved serine phosphorylation that occurs at the site of communication between the nucleotide-binding and substrate-binding domains of Hsp70. They found that phosphomimetic substitutions at this site dramatically downregulated heat shock responses and relocalization in response to starvation in budding yeast and in mammalian cells and blocked refolding of denatured proteins in vitro. Despite these negative effects, the phosphomimetic allele promoted survival of heavy metals and suppressed the [PSI+] prion phenotype in yeast, consistent with proposed roles for Hsp70 in generating self-nucleating seeds of misfolded proteins.

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

1H, 13C, 15N resonance assignments and secondary structure of yeast oligosaccharyltransferase subunit Ost4 and its functionally important mutant Ost4V23D

Asparagine-linked glycosylation is an essential and highly conserved protein modification reaction that occurs in the endoplasmic reticulum of cells during protein synthesis at the ribosome. In the central reaction, a pre-assembled high-mannose sugar is transferred from a lipid-linked donor substrate to the side-chain of an asparagine residue in an -N-X-T/S- sequence (where X is any residue except proline). This reaction is carried by a membrane-bound multi-subunit enzyme complex, oligosaccharyltransferase (OST). In humans, genetic defects in OST lead to a group of rare metabolic diseases collectively known as Congenital Disorders of Glycosylation. Certain mutations are lethal for all organisms. In yeast, the OST is composed of nine non-identical protein subunits. The functional enzyme complex contains eight subunits with either Ost3 or Ost6 at any given time. Ost4, an unusually small protein, plays a very important role in the stabilization of the OST complex. It bridges the catalytic subunit Stt3 with Ost3 (or Ost6) in the Stt3-Ost4-Ost3 (or Ost6) sub-complex. Mutation of any residue from M18-I24 in the trans-membrane helix of yeast Ost4 negatively impacts N-linked glycosylation and the growth of yeast. Indeed, mutation of valine23 to an aspartate impairs OST function in vivo resulting in a lethal phenotype in yeast. To understand the structural mechanism of Ost4 in the stabilization of the enzyme complex, we have initiated a detailed investigation of Ost4 and its functionally important mutant, Ost4V23D. Here, we report the backbone 1H, 13C, and 15N resonance assignments for Ost4 and Ost4V23D in dodecylphosphocholine micelles.

Blending wine yeast phenotypes with the aid of CRISPR DNA editing technologies

In recent years, CRISPR/Cas9-based genetic editing has become a mainstay in many laboratories including manipulations done with yeast. We utilized this technique to generate a self-cloned wine yeast strain that overexpresses two genes of oenological relevance i.e. the glycerol-3-phosphate dehydrogenase 1 (GPD1) and the alcohol acetyltransferase 1 (ATF1) directly implicated in glycerol and acetate ester production respectively. Riesling wine made from the resulting strain showed increased glycerol and acetate ester levels compared to the parental strain. In addition, significantly less acetic acid levels were measured in wine made with yeast containing both genetic alterations compared to wine made with the strain that only overexpresses GPD1. Thus, this strain provides an alternative strategy for alleviating the accumulation of acetic acid once glycerol production is favoured during alcoholic fermentation with the addition of dramatically increasing acetate esters production.

Multicellular group formation in Saccharomyces cerevisiae.

Understanding how and why cells cooperate to form multicellular organisms is a central aim of evolutionary biology. Multicellular groups can form through clonal development (where daughter cells stick to mother cells after division) or by aggregation (where cells aggregate to form groups). These different ways of forming groups directly affect relatedness between individual cells, which in turn can influence the degree of cooperation and conflict within the multicellular group. It is hard to study the evolution of multicellularity by focusing only on obligately multicellular organisms, like complex animals and plants, because the factors that favour multicellular cooperation cannot be disentangled, as cells cannot survive and reproduce independently. We support the use of Saccharomyces cerevisiae as an ideal model for studying the very first stages of the evolution of multicellularity. This is because it can form multicellular groups both clonally and through aggregation and uses a family of proteins called ‘flocculins’ that determine the way in which groups form, making it particularly amenable to laboratory experiments. We briefly review current knowledge about multicellularity in S. cerevisiae and then propose a framework for making predictions about the evolution of multicellular phenotypes in yeast based on social evolution theory. We finish by explaining how S. cerevisiae is a particularly useful experimental model for the analysis of open questions concerning multicellularity.

Biallelic variants in CTU2 cause DREAM-PL syndrome and impair thiolation of tRNA wobble U34.

The wobble position in the anticodon loop of transfer ribonucleic acid (tRNA) is subject to numerous posttranscriptional modifications. In particular, thiolation of the wobble uridine has been shown to play an important role in codon-anticodon interactions. This modification is catalyzed by a highly conserved CTU1/CTU2 complex, disruption of which has been shown to cause abnormal phenotypes in yeast, worms, and plants. We have previously suggested that a single founder splicing variant in human CTU2 causes a novel multiple congenital anomalies syndrome consisting of dysmorphic facies, renal agenesis, ambiguous genitalia, microcephaly, polydactyly, and lissencephaly (DREAM-PL). In this study, we describe five new patients with DREAM-PL phenotype and whose molecular analysis expands the allelic heterogeneity of the syndrome to five different alleles; four of which predict protein truncation. Functional characterization using patient-derived cells for each of these alleles, as well as the original founder allele; revealed a specific impairment of wobble uridine thiolation in all known thiol-containing tRNAs. Our data establish a recognizable CTU2-linked autosomal recessive syndrome in humans characterized by defective thiolation of the wobble uridine. The potential deleterious consequences for the translational efficiency and fidelity during development as a mechanism for pathogenicity represent an attractive target of future investigations.

PROTEIN RICH BIOMASS PRODUCTION BY NOVEL YEAST Cyberlindnera jadinii MMS7 USING Chara sp. HYDROLYSATE AND EVALUATION OF ITS ANTI-BACTERIAL ACTIVITY

A total of seven cultivable purified yeast phenotypes were isolated from soil collected from sugarecane bagasse dumping sites. By the genotypic characterization, new isolate MMS7 was confirmed as most promising yeast strain and it was identified as Cyberlindnera jadinii MMS7. We used this strain to investigate the feasibility of biomass production using hydrolysate of green Chara sp as sole medium. The ultrasonic pretreatment of the Chara sp biomass for hydrolyaste preparation was optimized as 0.35 WmL-1 (ultrasound power density) and 15 min (treatment time) using response surface methodology (RSM). The biomass of 15.05±0.12 g dwL-1 and protein concentration of 59.21±0.12% were obtained in C. jadinii MMS7, when cultivated in hydrolyaste. This study demonstrates the novel yeast C .jadinii MMS7 could be considered a promising single cell protein producing strain using Chara sp hydrolysate and it has high antibacterial activity against Bacillus cereus. Further, the exploitation of Chara sp. hydrolysate potential may used to aggregate value for algae cultivation. Keywords: Biomass, Cyberlindnera jadinii, Hydrolysate, Pretreatment, Chara sp

Life span extension of model yeast Saccharomyces cerevisiae upon ethanol derived-clover bud extract treatment

Antioxidant properties of clove bud have been widely studied due to its potential use in the pharmaceuticals field. One of the chemical bioactive compounds that show antioxidant activity is flavonoid. Our study revealed that the flavonoid content of ethanol-derived extract of clove bud was approximately 93.245 mg QE/100 gram. Less is known regarding the mode of actions of antioxidant from clove bud in cellular systems. In this study, we used model organism yeast Saccharomyces cerevisiae to study the action of antioxidant activity in cellular systems. We found that ethanol-derived clove bud extract (100 ppm) enhanced cells viability following H2O2-induced oxidative stress. Interestingly, clove bud extract increased yeast-antioxidative stress tolerance phenotype in a dose-independent manner. Suggesting, prooxidant activity of clove bud extract. Mitochondria have been known to involve in oxidative stress tolerance mechanisms primarily via mitochondrial adaptive ROS-signaling. Our data revealed that yeast mitochondrial membrane potential was unchanged following 100 ppm extract treatment yet significantly increased in higher extract treatment. Our study indicated that 100 ppm extract-supplementation in yeast culture resulted in a higher survival rate of yeast after 15-days of incubation, compared to that without extract treatments. We suggest that clove bud extract (100 ppm) could enhance oxidative stress tolerance phenotype in yeast S. cerevisiae, which then attributed on life span extension through its ROS scavenging activity. Further study must be conducted to confirm the underlying mechanisms of clove bud extract both physiologically and genetically.

Stress tolerance phenotype of industrial yeast: industrial cases, cellular changes, and improvement strategies.

Yeast is widely used in the baking, biocontrol, brewing, and bio manufacturing industries. In the baking industry alone, around two million tons of yeast are consumed worldwide every year. While yeast brings delicious and healthy lives to humans, we find that stress resistance of yeast is essential for the development of bioindustry. Whether during baking, biocontrol, brewing, bio manufacturing, or in other industries, yeast faces a variety of environmental stresses that have a great impact on its activity, transformation ability, etc., which make the production process uncertain. Therefore, robust yeast strains that can resist various environmental and endogenous stresses are needed. In recent years, many studies have investigated the stress resistance of laboratory strains and specific methods to improve stress resistance; however, applying these findings to industrial yeast is difficult. In this paper, based on summarizing the work of predecessors, we put forward the main steps to improve the stress resistance of industrial yeast systematically, which may provide a reference for researchers.

Characterization of the Copper Transporters from Lotus spp. and Their Involvement under Flooding Conditions.

Forage legumes are an important livestock nutritional resource, which includes essential metals, such as copper. Particularly, the high prevalence of hypocuprosis causes important economic losses to Argentinian cattle agrosystems. Copper deficiency in cattle is partially due to its low content in forage produced by natural grassland, and is exacerbated by flooding conditions. Previous results indicated that incorporation of Lotus spp. into natural grassland increases forage nutritional quality, including higher copper levels. However, the biological processes and molecular mechanisms involved in copper uptake by Lotus spp. remain poorly understood. Here, we identify four genes that encode putative members of the Lotus copper transporter family, denoted COPT in higher plants. A heterologous functional complementation assay of the Saccharomyces cerevisiae ctr1∆ctr3∆ strain, which lacks the corresponding yeast copper transporters, with the putative Lotus COPT proteins shows a partial rescue of the yeast phenotypes in restrictive media. Under partial submergence conditions, the copper content of L. japonicus plants decreases and the expression of two Lotus COPT genes is induced. These results strongly suggest that the Lotus COPT proteins identified in this work function in copper uptake. In addition, the fact that environmental conditions affect the expression of certain COPT genes supports their involvement in adaptive mechanisms and envisages putative biotechnological strategies to improve cattle copper nutrition.

Impact of Mutations in the DEXD‐motif of the Yeast RNA‐dependent ATPase Sub2

Pre‐mRNA splicing by the spliceosome is an essential part of eukaryotic gene expression. Errors in splicing can be detrimental to an organism, which is why it is extremely important to understand the spliceosome's mechanisms of assembly and action. Key features of splicing include the numerous RNA‐protein remodeling and RNA unwinding reactions that aid in spliceosome assembly. Some of these reactions require one of three, well‐conserved DEAD‐motif proteins that are part of the splicing machinery (Prp5/DDX46, Prp28/DDX23, and Sub2/UAP56). While Prp5 and Prp28 contain the canonical DEAD tetrapeptide motif within their active site, Sub2 is unusual in that it instead contains a conserved DECD motif. The goal of my research is to understand the function of the cysteine within Sub2's DECD motif in vitro and in vivo. Contrary to previous reports, I have shown that yeast containing a DECD to DEAD mutation in Sub2 are viable with little impact on yeast proliferation. To explain the conservation of Sub2's DECD sequence, we looked for changes in yeast phenotypes under stress conditions. I exposed yeast containing either DEAD‐Sub2 or DECD‐Sub2 to hydrogen peroxide and found that yeast containing DECD‐Sub2 died at lower oxidative stress conditions than the DEAD mutant. This suggests that Sub2's DECD‐motif cysteine is capable of being oxidized and that this results in a disruption in gene expression. Since one cysteine oxidation product (sulfinic acid) is isosteric with aspartate, I am now testing a DEDD mutant of Sub2 for function as well as attempting to chemically characterize the amino acid modifications resulting from Sub2 oxidation. Together my results will provide insight into a potential function for Sub2's conserved DECD‐motif cysteine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ergosterol Turnover in Yeast: An Interplay between Biosynthesis and Transport.

Sterols are important components of biological membranes that determine the physicochemical properties of lipid bilayer and regulate the functioning of membrane proteins. Being insoluble in water, sterols cannot diffuse between the membrane compartments separated by an aqueous phase. For this reason, distribution of sterols across cellular membranes is rather uneven. Membrane-to-membrane transport of sterols occurs mainly in a non-vesicular fashion and is provided by Lam and Osh proteins. In this review, we discuss the consequences of impairments in sterol biosynthesis and transport mostly relying on the studies performed on the model organism Saccharomyces cerevisiae. Despite the fact that molecular mechanisms underlying the functioning of Lam and Osh proteins are well established, the biological roles of these proteins are still unclear, because deletions of corresponding genes do not affect yeast phenotype. At the same time, disruptions in the biosynthesis of ergosterol, the major sterol of S. cerevisiae, lead to either cell death or reduced stress resistance. However, under certain conditions (e.g., mild salt or thermal stresses), a decrease in the ergosterol levels causes an increase in cell resistance. This suggests that the cells possess a mechanism facilitating rapid adjustment of the plasma membrane sterol content. We argue that the biological role of Lam proteins is, in particular, fast optimization of sterol composition of cell membranes.

Identification, design and synthesis of novel pyrazolopyridine influenza virus nonstructural protein 1 antagonists

Nonstructural protein 1 (NS1) plays a crucial function in the replication, spread, and pathogenesis of influenza virus by inhibiting the host innate immune response. Here we report the discovery and optimization of novel pyrazolopyridine NS1 antagonists that can potently inhibit influenza A/PR/8/34 replication in MDCK cells, rescue MDCK cells from cytopathic effects of seasonal influenza A strains, reverse NS1-dependent inhibition of IFN-β gene expression, and suppress the slow growth phenotype in NS1-expressing yeast. These pyrazolopyridines will enable researchers to investigate NS1 function during infection and how antagonists can be utilized in the next generation of treatments for influenza infection.

Coupling of Human Rhodopsin to a Yeast Signaling Pathway Enables Characterization of Mutations Associated with Retinal Disease.

G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in eukaryotes, with multiple GPCR mutations linked to human diseases. With the growing number of sequenced human genomes, determining the pathogenicity of a mutation is challenging, but can be aided by a direct measurement of GPCR-mediated signaling. This is particularly difficult for the visual pigment rhodopsin-a GPCR activated by light-for which hundreds of mutations have been linked to inherited degenerative retinal diseases such as retinitis pigmentosa. In this study, we successfully engineered, for the first time, activation by human rhodopsin of the yeast mating pathway, resulting in signaling via a fluorescent reporter. We combine this novel assay for rhodopsin light-dependent activation with studies of subcellular localization, and the upregulation of the unfolded protein response in response to misfolded rhodopsin protein. We use these assays to characterize a panel of rhodopsin mutations with known molecular phenotypes, finding that rhodopsin maintains a similar molecular phenotype in yeast, with some interesting differences. Furthermore, we compare our assays in yeast with clinical phenotypes from patients with novel disease-linked mutations. We demonstrate that our engineered yeast strain can be useful in rhodopsin mutant classification, and in helping to determine the molecular mechanisms underlying their pathogenicity. This approach may also be applied to better understand the clinical relevance of other human GPCR mutations, furthering the use of yeast as a tool for investigating molecular mechanisms relevant to human disease.

The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin

Bacterial nucleotidyl cyclase toxins are potent virulence factors that upon entry into eukaryotic cells are stimulated by endogenous cofactors to catalyze the production of large amounts of 3'5'-cyclic nucleoside monophosphates. The activity of the effector ExoY from Pseudomonas aeruginosa is stimulated by the filamentous form of actin (F-actin). Utilizing yeast phenotype analysis, site-directed mutagenesis, functional biochemical assays, and confocal microscopy, we demonstrate that the last nine amino acids of the C terminus of ExoY are crucial for the interaction with F-actin and, consequently, for ExoY's enzymatic activity in vitro and toxicity in a yeast model. We observed that isolated C-terminal sequences of P. aeruginosa ExoY that had been fused to a carrier protein bind to F-actin and that synthetic peptides corresponding to the extreme ExoY C terminus inhibit ExoY enzymatic activity in vitro and compete with the full-length enzyme for F-actin binding. Interestingly, we noted that various P. aeruginosa isolates of the PA14 family, including highly virulent strains, harbor ExoY variants with a mutation altering the C terminus of this effector. We found that these naturally occurring ExoY variants display drastically reduced enzymatic activity and toxicity. Our findings shed light on the molecular basis of the ExoY-F-actin interaction, revealing that the extreme C terminus of ExoY is critical for binding to F-actin in target cells and that some P. aeruginosa isolates carry C-terminally mutated, low-activity ExoY variants.

Filamentation in Candida auris, an emerging fungal pathogen of humans: passage through the mammalian body induces a heritable phenotypic switch

Morphological plasticity has historically been an indicator of increased virulence among fungal pathogens, allowing rapid adaptation to changing environments. Candida auris has been identified as an emerging multidrug-resistant human pathogen of global importance. Since the discovery of this species, it has been thought that C. auris is incapable of filamentous growth. Here, we report the discovery of filamentation and three distinct cell types in C. auris: typical yeast, filamentation-competent (FC) yeast, and filamentous cells. These cell types form a novel phenotypic switching system that contains a heritable (typical yeast-filament) and a nonheritable (FC-filament) switch. Intriguingly, the heritable switch between the typical yeast and the FC/filamentous phenotype is triggered by passage through a mammalian body, whereas the switch between the FC and filamentous phenotype is nonheritable and temperature-dependent. Low temperatures favor the filamentous phenotype, whereas high temperatures promote the FC yeast phenotype. Systemic in vivo and in vitro investigations were used to characterize phenotype-specific variations in global gene expression, secreted aspartyl proteinase (SAP) activity, and changes in virulence, indicating potential for niche-specific adaptations. Taken together, our study not only sheds light on the pathogenesis and biology of C. auris but also provides a novel example of morphological and epigenetic switching in fungi.

Genetic Variation in a Cellular Adhesin Suggests Self-Discrimination Driven by Ecological Competition in Yeast

Kin selection can explain most examples of altruism, with kin recognition common in many animal systems. In microbes, cooperative behaviors involving cell-cell adherence generally rely on “kind” recognition, in which a greenbeard locus signals cooperation regardless of overall relatedness. However, in motile microbes that must locate one another and avoid cheaters, rare examples of kin- and self-recognition have been reported; variable membrane-associated proteins confer discrimination. In contrast, clonal growth of non-motile microbes generates patches of highly-related individuals, thus obviating the need for discriminatory systems. Instead, kind-recognition adhesion can serve as an adaptive strategy for a lineage in ecological competition. Here we use population genetic data to investigate Flo11, a membrane-anchored adhesion protein required for social phenotypes in the budding yeast, and find evidence that it functions in both ways. Surprisingly, the regions implicated in cell-cell interaction exhibit a signature of positive selection, suggesting the possibility of a recognition system in which cells preferentially interact with like kinds. At an upstream activation site, there is evidence for balancing selection, thus suggesting selection on variegated gene expression, which has been proposed as an adaptive strategy in clonal yeast populations. Phenotypic assays support these inferences and demonstrate that different alleles confer a competitive advantage. Unlike in motile microbes where cheater avoidance is driving the evolution of recognition, competition among clones may be the evolution of homophilic binding in S. cerevisiae. Thus, the interplay between inter-clone competition and intra-clone cooperation in spatially structured microbial communities may lead to complex recognition systems.

Generalizing a mathematical model of prion aggregation allows strain coexistence and co-stability by including a novel misfolded species

Prions are proteins capable of adopting misfolded conformations and transmitting these conformations to other normally folded proteins. Prions are most commonly known for causing fatal neurodegenerative diseases in mammals but are also associated with several harmless phenotypes in yeast. A distinct feature of prion propagation is the existence of different phenotypical variants, called strains. It is widely accepted that these strains correspond to different conformational states of the protein, but the mechanisms driving their interactions remain poorly understood. This study uses mathematical modeling to provide insight into this problem. We show that the classical model of prion dynamics allows at most one conformational strain to stably propagate. In order to conform to biological observations of strain coexistence and co-stability, we develop an extension of the classical model by introducing a novel prion species consistent with biological studies. Qualitative analysis of this model reveals a new variety of behavior. Indeed, it allows for stable coexistence of different strains in a wide parameter range, and it also introduces intricate initial condition dependency. These new behaviors are consistent with experimental observations of prions in both mammals and yeast. As such, our model provides a valuable tool for investigating the underlying mechanisms of prion propagation and the link between prion strains and strain specific phenotypes. The consideration of a novel prion species brings a change in perspective on prion biology and we use our model to generate hypotheses about prion infectivity.

Carnitine Requires Choline to Exert Physiological Effects in Saccharomyces cerevisiae.

L-Carnitine is a key metabolite in the energy metabolism of eukaryotic cells, functioning as a shuttling molecule for activated acyl-residues between cellular compartments. In higher eukaryotes this function is essential, and defects in carnitine metabolism has severe effects on fatty acid and carbon metabolism. Carnitine supplementation has been associated with an array of mostly beneficial impacts in higher eukaryotic cells, including stress protection and regulation of redox metabolism in diseased cells. Some of these phenotypes have no obvious link to the carnitine shuttle, and suggest that carnitine has as yet unknown shuttle-independent functions. The existence of shuttle-independent functions has also been suggested in Saccharomyces cerevisiae, including a beneficial effect during hydrogen peroxide stress and a detrimental impact when carnitine is co-supplemented with the reducing agent dithiothreitol (DTT). Here we used these two distinct yeast phenotypes to screen for potential genetic factors that suppress the shuttle independent physiological effects of carnitine. Two deletion strains, Δcho2 and Δopi3, coding for enzymes that catalyze the sequential conversion of phosphatidylethanolamine to phosphatidylcholine were identified for suppressing the phenotypic effects of carnitine. Additional characterisation indicated that the suppression cannot be explained by differences in phospholipid homeostasis. The phenotypes could be reinstated by addition of extracellular choline, but show that the requirement for choline is not based on some overlapping function or the structural similarities of the two molecules. This is the first study to suggest a molecular link between a specific metabolite and carnitine-dependent, but shuttle-independent phenotypes in eukaryotes.