Cardiolipin (CL) is a unique lipid component of mitochondria in all eukaryotes. It is important for the architecture of mitochondrial membranes and for mitochondrial dynamics. CL also creates a ...highly specific microenvironment of mitochondrial protein machineries. CL biosynthetic pathway is, however, only partially characterized in the fission yeast Schizosaccharomyces pombe. Here we show that CL synthase is an essential protein in S. pombe. It is encoded by the ORF SPAC22A12.08c as a C terminal part of a tandem fusion protein together with a mitochondrial hydrolase of unknown function. Expression of S. pombe CL synthase is able to complement deletion of the CRD1 gene of Saccharomyces cerevisiae and, vice versa, S. cerevisiae CRD1 gene complements deletion of S. pombe SPAC22A12.08c. The proper expression of CL synthase and its partner in the tandem protein, the mitochondrial hydrolase, is regulated at the level of alternate intron splicing. The first part of the SPAC22A12.08c fusion protein could be translated from both major SPAC22A12.08c derived mRNAs, with and without intron IV. Functional CL synthase, however, is produced only from the minor SPAC22A12.08c derived mRNA that has intron IV retained. Thus, intron retention is a novel mechanism for the differential expression of two proteins that evolved as a fusion protein and are under the control of the same promoter.
•Cardiolipin synthase is essential in the yeast Schizosaccharomyces pombe.•S. pombe CL synthase is part of a tandem fusion protein encoded by SPAC22A12.08c.•SPAC22A12.08c sequence is 258 bp longer than annotated and contains 7 introns.•Expression of parts of SPAC22A12.08c fusion protein is ensured by intron retention.•CL synthase is produced only from SPAC22A12.08c mRNA in which intron IV is retained.
Mitochondrial DNA (mtDNA) molecules are packaged into compact nucleo-protein structures called mitochondrial nucleoids (mt-nucleoids). Their compaction is mediated in part by high-mobility group ...(HMG)-box containing proteins (mtHMG proteins), whose additional roles include the protection of mtDNA against damage, the regulation of gene expression and the segregation of mtDNA into daughter organelles. The molecular mechanisms underlying these functions have been identified through extensive biochemical, genetic, and structural studies, particularly on yeast (Abf2) and mammalian mitochondrial transcription factor A (TFAM) mtHMG proteins. The aim of this paper is to provide a comprehensive overview of the biochemical properties of mtHMG proteins, the structural basis of their interaction with DNA, their roles in various mtDNA transactions, and the evolutionary trajectories leading to their rapid diversification. We also describe how defects in the maintenance of mtDNA in cells with dysfunctional mtHMG proteins lead to different pathologies at the cellular and organismal level.
CRISPR-Cas9 is a genome-editing technique that has been widely adopted thanks to its simplicity, efficiency, and broad application potential. Due to its advantages and pervasive use, there have been ...attempts to include this method in the existing curricula for students majoring in various disciplines of biology. In this perspective, we summarize the existing CRISPR-Cas courses that harness a well-established model organism: baker's yeast,
. As an example, we present a detailed description of a fully hands-on, flexible, robust, and cost-efficient practical CRISPR-Cas9 course, where students participate in yeast genome editing at every stage-from the bioinformatic design of single-guide RNA, through molecular cloning and yeast transformation, to the final confirmation of the introduced mutation. Finally, we emphasize that in addition to providing experimental skills and theoretical knowledge, the practical courses on CRISPR-Cas represent ideal platforms for discussing the ethical implications of the democratization of biology.
The biosynthesis of yeast phosphatidylglycerol (PG) takes place in the inner mitochondrial membrane. Outside mitochondria, the abundance of PG is low. Here, we present evidence that the subcellular ...distribution of PG is maintained by the locally controlled enzymatic activity of the PG-specific phospholipase, Pgc1. A fluorescently labeled Pgc1 protein accumulates on the surface of lipid droplets (LD). We show, however, that LD are not only dispensable for Pgc1-mediated PG degradation, but do not even host any phospholipase activity of Pgc1. Our in vitro assays document the capability of LD-accumulated Pgc1 to degrade PG upon entry to the membranes of the endoplasmic reticulum, mitochondria and even of artificial phospholipid vesicles. Fluorescence recovery after photobleaching analysis confirms the continuous exchange of GFP-Pgc1 within the individual LD in situ, suggesting that a steady-state equilibrium exists between LD and membranes to regulate the immediate phospholipase activity of Pgc1. In this model, LD serve as a storage place and shelter Pgc1, preventing its untimely degradation, while both phospholipase activity and degradation of the enzyme occur in the membranes.
The yeast
Magnusiomyces capitatus
is an opportunistic human pathogen causing rare yet severe infections, especially in patients with hematological malignancies. Here, we report the 20.2 megabase ...genome sequence of an environmental strain of this species as well as the genome sequences of eight additional isolates from human and animal sources providing an insight into intraspecies variation. The distribution of single-nucleotide variants is indicative of genetic recombination events, supporting evidence for sexual reproduction in this heterothallic yeast. Using RNAseq-aided annotation, we identified genes for 6518 proteins including several expanded families such as kexin proteases and Hsp70 molecular chaperones. Several of these families are potentially associated with the ability of
M. capitatus
to infect and colonize humans. For the purpose of comparative analysis, we also determined the genome sequence of a closely related yeast,
Magnusiomyces ingens
. The genome sequences of
M. capitatus
and
M. ingens
exhibit many distinct features and represent a basis for further comparative and functional studies.
The PEL1/PGS1 gene of the yeast Saccharomyces cerevisiae is essential for the viability of rho-/rho degrees mutants and the normal cardiolipin content of cells. The PEL1-GFP fusion gene has been ...found to complement the pel1/pgs1 mutation and its fluorescent protein was localized to mitochondria similarly to the beta-galactosidase activity of a protein encoded by the PEL1-lacZ fusion gene. The expression of the PEL1-lacZ reporter gene was repressed in cells grown in the presence of inositol and choline, reduced in the ino2 and ino4 strains, but constitutive in the opi1 null-mutant strain. The results demonstrate that Pel1p, playing a vital role in cells impaired in the mitochondrial DNA, is localized in the mitochondria and expressed in response to inositol and choline.
Ionophores such as valinomycin and nigericin are potent tools for studying the impact of ion perturbance on cellular functions. To obtain a broader picture about molecular components involved in ...mediating the effects of these drugs on yeast cells under respiratory growth conditions, we performed a screening of the haploid deletion mutant library covering the Saccharomyces cerevisiae nonessential genes. We identified nearly 130 genes whose absence leads either to resistance or to hypersensitivity to valinomycin and/or nigericin. The processes affected by their protein products range from mitochondrial functions through ribosome biogenesis and telomere maintenance to vacuolar biogenesis and stress response. Comparison of the results with independent screenings performed by our and other laboratories demonstrates that although mitochondria might represent the main target for both ionophores, cellular response to the drugs is very complex and involves an intricate network of proteins connecting mitochondria, vacuoles, and other membrane compartments.
CTBT (7-chlorotetrazolo5,1-cbenzo1,2,4triazine) is an antifungal and chemosensitizing agent that induces oxidative stress in yeast and filamentous fungi and enhances the cytotoxic activity of ...5-fluorocytosine and azole antimycotics. This study reports the effect of CTBT on bacterial cells. CTBT inhibited the growth of both Gram-positive and Gram-negative bacterial species. The action of CTBT was bactericidal. In Escherichia coli, CTBT induced an increased formation of reactive oxygen species (ROS), as determined with a ROS specific probe 2′,7′-dichlorodihydrofluorescein diacetate. In zone inhibition assays, bacterial cells were more sensitive to CTBT compared with paraquat, menadione and hydrogen peroxide. The deletion of oxidative stress related genes resulted in increased susceptibility of E. coli mutant strains to CTBT treatment. Exogenous antioxidants such as ascorbic acid, cysteine and glutathione exhibited a protective effect against the growth inhibition induced by CTBT. CTBT may be a useful tool in the studies of ROS generation, oxidant sensing and oxidative stress response in different bacterial species.
Rhodamine 6G is a highly fluorescent dye often used to determine the transport activity of yeast membrane efflux pumps. The ATP-binding cassette transporter
Pdr5p confers resistance to several ...unrelated drugs in
Pdr5p also extrudes rhodamine 6G (R6G) from intact yeast cells in an energy-dependent manner. Incubation of yeast cells in the presence of 2-deoxy-D-glucose (inhibitor of glycolysis) and R6G (mitochondrial ATPase inhibitor) leads to marked depletion of intracellular ATP pool ( Kolaczkowski
, 1996 ). An active
Pdr5p mediated extrusion of R6G from intact yeast cells can be followed by direct measurement of the fluorescence of extruded R6G in the assay buffer.
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