SUMMARY Mitochondrial morphology is maintained by the opposing activities of dynamin-based fission and fusion machines. In response to stress, this balance is dramatically shifted toward fission. This study reveals that the yeast transcriptional repressor cyclin C is both necessary and sufficient for stress-induced hyper-fission. In response to oxidative stress, cyclin C translocates from the nucleus to the cytoplasm where it is destroyed. Prior to its destruction, cyclin C both genetically and physically interacts with Mdv1p, an adaptor that links the GTPase Dnm1p to the mitochondrial receptor Fis1p. Cyclin C is required for stress-induced Mdv1p mitochondrial recruitment and the efficient formation of functional Dnm1p filaments. Finally, co-immunoprecipitation studies and fluorescence microscopy revealed an elevated association between Mdv1p and Dnm1p in stressed cells that is dependent on cyclin C. This study provides a mechanism by which stress-induced gene induction and mitochondrial fission are coordinated through translocation of cyclin C.
In response to oxidative stress, cyclin C translocates from the nucleus to the cytoplasm, where it interacts with the mitochondrial fission machinery and induces extensive fragmentation of this organelle. Med13p is identified as the anchor protein that retains cyclin C in the nucleus.
Resistance to azole drugs, the major clinical antifungal compounds, is most commonly due to gain-of-function (GOF) substitution mutations in a gene called PDR1 in the fungal pathogen Candida glabrata. PDR1 encodes a zinc cluster-containing transcription factor. GOF forms of Pdr1 drive high level expression of downstream target gene expression with accompanying azole resistance. PDR1 has two homologous genes in Saccharomyces cerevisiae, called ScPDR1 and ScPDR3. This study provides evidence that the PDR1 gene in C. glabrata represents a blend of the properties found in the two S. cerevisiae genes. We demonstrated that GOF Pdr1 derivatives are overproduced at the protein level and less stable than the wild-type protein. Overproduction of wild-type Pdr1 increased target gene expression but to a lesser extent than GOF derivatives. Site-directed mutagenesis of Pdr1 binding sites in the PDR1 promoter provided clear demonstration that autoregulation of PDR1 is required for its normal function. An internal deletion mutant of Pdr1 lacking its central regulatory domain behaved as a hyperactive transcription factor that was lethal unless conditionally expressed. A full understanding of the regulation of Pdr1 will provide a new avenue of interfering with azole resistance in C. glabrata.
h ATP-binding cassette transporters Pdr5 and Yor1 from Saccharomyces cerevisiae control the asymmetric distribution of phospholipids across the plasma membrane as well as serving as ATP-dependent drug efflux pumps. Mutant strains lacking these transporter proteins were found to exhibit very different resistance phenotypes to two inhibitors of sphingolipid biosynthesis that act either late (aureobasidin A [AbA]) or early (myriocin [Myr]) in the pathway leading to production of these important plasma membrane lipids. These pdr5⌬ yor1 strains were highly AbA resistant but extremely sensitive to Myr. We provide evidence that these phenotypic changes are likely due to modulation of the plasma membrane flippase complexes, Dnf1/Lem3 and Dnf2/Lem3. Flippases act to move phospholipids from the outer to the inner leaflet of the plasma membrane. Genetic analyses indicate that lem3⌬ mutant strains are highly AbA sensitive and Myr resistant. These phenotypes are fully epistatic to those seen in pdr5⌬ yor1 strains. Direct analysis of AbA-induced signaling demonstrated that loss of Pdr5 and Yor1 inhibited the AbA-triggered phosphorylation of the AGC kinase Ypk1 and its substrate Orm1. Microarray experiments found that a pdr5⌬ yor1 strain induced a Pdr1-dependent induction of the entire Pdr regulon. Our data support the view that Pdr5/Yor1 negatively regulate flippase function and activity of the nuclear Pdr1 transcription factor. Together, these data argue that the interaction of the ABC transporters Pdr5 and Yor1 with the Lem3-dependent flippases regulates permeability of AbA via control of plasma membrane protein function as seen for the high-affinity tryptophan permease Tat2.
Chromatin modification is traditionally assessed in biochemical assays that provide average measurements of static events given that the analysis requires components from many cells. Microscopy can visualize single cells, but the cell body and organelles can hamper staining and visualization of the nucleus. Normally, chromatin is visualized by immunostaining a fixed sample or by expressing exogenous fluorescently tagged proteins in a live cell. Alternative microscopy tools to observe changes of endogenous chromatin in real-time are needed. Here, we isolated transcriptionally competent nuclei from cells and used antibody staining without fixation to visualize changes in endogenous chromatin. This method allows the real-time addition of drugs and fluorescent probes to one or more nuclei while under microscopy observation. A high-resolution map of 11 endogenous nuclear markers of the histone code, transcription machinery and architecture was obtained in transcriptionally active nuclei by performing confocal and structured illumination microscopy. We detected changes in chromatin modification and localization at the single-nucleus level after inhibition of histone deacetylation. Applications in the study of RNA transcription, viral protein function and nuclear architecture are presented. This article has an associated First Person interview with the first author of the paper.
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