Getting a Transcription Factor to Only One Nucleus Following Mitosis

The Ace2 transcription factor from budding yeast has both a regulated nuclear localization signal and a regulated nuclear export signal, and Ace2 phosphorylation by the Cbk1 kinase results in Ace2 accumulation in daughter cells but not mothers

The yeast Cbk1 kinase regulates mRNA localization via the mRNA-binding protein Ssd1

In the absence of Cbk1 phosphorylation Ssd1-associated mRNAs are redirected from sites of polarized cell growth to stress granules and P-bodies

A synthetic circuit for selectively arresting daughter cells to create aging populations

The ability to engineer genetic programs governing cell fate will permit new safeguards for engineered organisms and will further the biological understanding of differentiation and aging. Here, we have designed, built and implemented a genetic device in the budding yeast Saccharomyces cerevisiae that controls cell-cycle progression selectively in daughter cells. The synthetic device was built in a modular fashion by combining timing elements that are coupled to the cell cycle, i.e. cell-cycle specific promoters and protein degradation domains, and an enzymatic domain which conditionally confers cell arrest. Thus, in the presence of a drug, the device is designed to arrest growth of only newly-divided daughter cells in the population. Indeed, while the engineered cells grow normally in the absence of drug, with the drug the engineered cells display reduced, linear growth on the population level. Fluorescence microscopy of single cells shows that the device induces cell arrest exclusively in daughter cells and radically shifts the age distribution of the resulting population towards older cells. This device, termed the ‘daughter arrester’, provides a blueprint for more advanced devices that mimic developmental processes by having control over cell growth and death

Sphingolipid accumulation causes mitochondrial dysregulation and cell death

Sphingolipids are structural components of cell membranes that have signaling roles to regulate many activities, including mitochondrial function and cell death. Sphingolipid metabolism is integrated with numerous metabolic networks, and dysregulated sphingolipid metabolism is associated with disease. Here, we describe a monogenic yeast model for sphingolipid accumulation. A csg2Δ mutant cannot readily metabolize and accumulates the complex sphingolipid inositol phosphorylceramide (IPC). In these cells, aberrant activation of Ras GTPase is IPC-dependent, and accompanied by increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial mass. Survival or death of csg2Δ cells depends on nutritional status. Abnormal Ras activation in csg2Δ cells is associated with impaired Snf1/AMPK protein kinase, a key regulator of energy homeostasis. csg2Δ cells are rescued from ROS production and death by overexpression of mitochondrial catalase Cta1, abrogation of Ras hyperactivity or genetic activation of Snf1/AMPK. These results suggest that sphingolipid dysregulation compromises metabolic integrity via Ras and Snf1/AMPK pathways

Human mitochondrial cytochrome c oxidase assembly factor COX18 acts transiently as a membrane insertase within the subunit 2 maturation module

Defects in mitochondrial cytochrome c oxidase or respiratory chain complex IV (CIV) assembly are a frequent cause of human mitochondrial disorders. Specifically, mutations in four conserved assembly factors impinging the biogenesis of the mitochondrion-encoded catalytic core subunit 2 (COX2) result in myopathies. These factors afford stability of newly synthesized COX2 (the dystonia-ataxia syndrome protein COX20), a protein with two transmembrane domains, and maturation of its copper center, CuA (cardiomyopathy proteins SCO1, SCO2, and COA6). COX18 is an additional COX2 assembly factor that belongs to the Oxa1 family of membrane protein insertases. Here, we used a gene-editing approach to generate a human COX18 knock-out HEK293T cell line that displays isolated complete CIV deficiency. We demonstrate that COX20 stabilizes COX2 during insertion of its N-proximal transmembrane domain, and subsequently, COX18 transiently interacts with COX2 to promote translocation across the inner membrane of the COX2 C-tail that contains the apo-CuA site. The release of COX18 from this complex coincides with the binding of the SCO1-SCO2-COA6 copper metallation module to COX2-COX20 to finalize COX2 biogenesis. Therefore, COX18 is a new candidate when screening for mitochondrial disorders associated with isolated CIV deficiency

A CMC1 -knockout reveals translation-independent control of human mitochondrial complex IV biogenesis

Defects in mitochondrial respiratory chain complex IV (CIV) frequently cause encephalocardiomyopathies. Human CIV assembly involves 14 subunits of dual genetic origin and multiple nucleus-encoded ancillary factors. Biogenesis of the mitochondrion-encoded copper/heme-containing COX1 subunit initiates the CIV assembly process. Here, we show that the intermembrane space twin CX C protein CMC1 forms an early CIV assembly intermediate with COX1 and two assembly factors, the cardiomyopathy proteins COA3 and COX14. A TALEN-mediated knockout HEK293T cell line displayed normal COX1 synthesis but decreased CIV activity owing to the instability of newly synthetized COX1. We demonstrate that CMC1 stabilizes a COX1-COA3-COX14 complex before the incorporation of COX4 and COX5a subunits. Additionally, we show that CMC1 acts independently of CIV assembly factors relevant to COX1 metallation (COX10, COX11, and SURF1) or late stability (MITRAC7). Furthermore, whereas human COX14 and COA3 have been proposed to affect COX1 mRNA translation, our data indicate that CMC1 regulates turnover of newly synthesized COX1 prior to and during COX1 maturation, without affecting the rate of COX1 synthesis

Rôle du complexe RAM et de la NDR kinase Cbk1p dans la séparation cellulaire, la polarité et la fertilité chez la levure Saccharomyces cerevisiae

La polarité cellulaire est un phénomène central dans tous les organismes. Elle organise la cellule en permettant notamment une croissance localisée à un site précis de la membrane, nettement visible dans les neurones. Chez la levure Saccharomyces cerevisiae la polarité est également importante lors de diverses événements cellulaires, la sélection du site de bourgeonnement, la croissance végétative avec ses cellules ovales, la croissance filamenteuse avec ses cellules allongées et au cours du croisement avec la formation d un long prolongement cytoplasmique, le shmoo permettant la formation d un zygote à partir de deux levures de signe sexuel opposé Mata et Mat . Chez S. cerevisiae les RAM (Regulation of Ace2p and cellular Morphogenesis) sont un ensemble de six gènes (CBK1, MOB2, HYM1, TAO3, KIC1 et SOG2), qui codent des protéines interagissant les unes avec les autres et localisées aux sites de croissance polarisée de la cellule ainsi que dans le noyau de la cellule fille pour Cbk1p et Mob2p. Les protéines RAM régulent au moins trois fonctions différentes : la séparation cellulaire, la viabilité cellulaire en interaction avec la protéine Ssd1p et la polarité cellulaire. Aucune relation claire entre les RAM et les composants importants connus comme impliqués dans la polarité n est connue. La sérine thréonine kinase Cbk1p appartient à la famille des NDR (Nuclear Dbf2 related) qui possède des homologues chez différents organismes et semble être l effecteur final de la voie RAM. Nous nous sommes donc focalisé sur l étude de cette protéine dont l activité kinase est régulée par la phosphorylation de la sérine 570 et de la thréonine 743.Cellular polarity is a central phenomena in all organisms, which structures the cell and allows growth at specific sites at the cell surface, as is clearly seen in neurons. In the yeast Saccharomyces cerevisiae, polarity is important for many processes: selection of the bud site, polarized vegetative growth to produce the characteristic ellipsoidal cells, filamentous growth with its elongated cells and the formation of the long projection, or shmoo, during mating, allowing the formation of zygotes from cells of opposite mating types, MAT a and MAT . In S. cerevisiae, the RAM network (Regulation of Ace2p and cellular Morphogenesis) is a group of six genes (CBK1, MOB2, HYM1, TAO3, KIC1 and SOG), which encode proteins that interact with each other and localize to the sites of polarized growth and in the case of Cbk1p and Mob2p, in the nucleus of the daughter cell just before cytokinesis. The RAM network regulates at least three different functions: cell separation after cytokinesis, cell viability in interaction with Ssd1p and cell polarity. To date, no connection has been described between the RAM proteins and other proteins known to be involved in establishing, or maintaining, cell polarity. Cbk1p is a serine threonine protein kinase of the NDR family (Nuclear Dbf2 Related) that is conserved amongst eukaryotes and appears to be the final effecter of the RAM network. We have focused our attention on the study of this protein, whose activity is regulated by the phosphorylation of at least two residues, serine 570 and threonine 743.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Redox and Reactive Oxygen Species Regulation of Mitochondrial Cytochrome c Oxidase Biogenesis

Significance: Cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is the major oxygen consumer enzyme in the cell. COX biogenesis involves several redox-regulated steps. The process is highly regulated to prevent the formation of pro-oxidant intermediates. Recent Advances: Regulation of COX assembly involves several reactive oxygen species and redox-regulated steps. These include: (i) Intricate redox-controlled machineries coordinate the expression of COX isoenzymes depending on the environmental oxygen concentration. (ii) COX is a heme A-copper metalloenzyme. COX copper metallation involves the copper chaperone Cox17 and several other recently described cysteine-rich proteins, which are oxidatively folded in the mitochondrial intermembrane space. Copper transfer to COX subunits 1 and 2 requires concomitant transfer of redox power. (iii) To avoid the accumulation of reactive assembly intermediates, COX is regulated at the translational level to minimize synthesis of the heme A-containing Cox1 subunit when assembly is impaired. Critical Issues: An increasing number of regulatory pathways converge to facilitate efficient COX assembly, thus preventing oxidative stress. Future Directions: Here we will review on the redox-regulated COX biogenesis steps and will discuss their physiological relevance. Forthcoming insights into the precise regulation of mitochondrial COX biogenesis in normal and stress conditions will likely open future perspectives for understanding mitochondrial redox regulation and prevention of oxidative stress. Antioxid. Redox Signal . 19, 1940–1952