mRNA translation in yeast during entry into stationary phase

The expression of some Saccharomyces cerevisiae genes is induced as cells enter stationary phase. Their mRNAs are translated during a period in the growth cycle when the translational apparatus is relatively inert, thereby raising the possibility that these mRNAs compete effectively for a limiting pool of translation factors. To test this idea, the translation of mRNAs carrying different 5′-leaders was compared during exponential growth and after entry into stationary phase upon glucose starvation. Closely related sets of lacZ mRNAs, carrying 5′-leaders from the PYK1, PGK1, RpL3, Rp29, HSP12, HSP26 or THI4 mRNAs, were studied. These mRNAs displayed differing translational efficiencies during exponential growth, but their relative translatabilities were not significantly affected by entry into stationary phase, indicating that they compete just as effectively under these conditions. Polysome analysis revealed that the wild-type PYK1, ACT1 and HSP26 mRNAs are all translated efficiently during stationary phase, when the translational apparatus is relatively inert. Also, significant levels of the translation initiation factors eIF-2α, eIF-4E and eIF-4A were maintained during the growth cycle. These data are consistent with the idea that, while translational activity decreases dramatically during entry into stationary phase, yeast cells maintain excess translational capacity under these conditions. Received: 31 March 1998 / Accepted: 4 May 1998     

Mitochondrial dysfunction increases oxidative stress and decreases chronological life span in fission yeast

Background

Oxidative stress is a probable cause of aging and associated diseases. Reactive oxygen species (ROS) originate mainly from endogenous sources, namely the mitochondria.

Methodology/Principal Findings

We analyzed the effect of aerobic metabolism on oxidative damage in Schizosaccharomyces pombe by global mapping of those genes that are required for growth on both respiratory-proficient media and hydrogen-peroxide-containing fermentable media. Out of a collection of approximately 2700 haploid yeast deletion mutants, 51 were sensitive to both conditions and 19 of these were related to mitochondrial function. Twelve deletion mutants lacked components of the electron transport chain. The growth defects of these mutants can be alleviated by the addition of antioxidants, which points to intrinsic oxidative stress as the origin of the phenotypes observed. These respiration-deficient mutants display elevated steady-state levels of ROS, probably due to enhanced electron leakage from their defective transport chains, which compromises the viability of chronologically-aged cells.

Conclusion/Significance

Individual mitochondrial dysfunctions have often been described as the cause of diseases or aging, and our global characterization emphasizes the primacy of oxidative stress in the etiology of such processes.     

Monitoring dynamics of gene expression in yeast during stationary phase.

The commonly used genetic approaches in yeast are designed to identify defects in cell/colony growth. In order to identify genes which control molecular mechanisms during quiescence ('stationary phase'), different tactics are required. We describe the development of a new genetic approach based on the previous observations that gene expression in quiescent Saccharomyces cerevisiae cells is largely repressed. For studying the mechanism controlling the repression of gene expression in stationary phase, we use UBI4-lacZ as a reporter gene. The product of this fusion gene was shown previously to encode an unstable protein in dividing cells. We show here that it is also unstable in stationary cells. We demonstrate that the relatively short half-life of this reporter protein can be utilized to monitor the dynamics of the repression of gene expression during stationary phase in liquid culture, using ACT1 or SSA3 promoters as the model promoters. By adapting a colony color test, we show that the reporter gene can also be used to monitor gene expression in quiescent colonies, thus serving as a tool to screen for defects in the regulation of this process during growth arrest. The utility of the approach was demonstrated by confirming the defects of top1Delta and bcy1Delta cells to appropriately express the ACT1p-UBI4-lacZ in stationary phase. The mutant colonies were easily discernible from wild-type colonies by our color test. Finally, using SSA3p-UBI4-lacZ as the reporter gene, we found that the 5'-untranslated region of SSA3 mRNA is sufficient to repress translation of the reporter mRNA after entry of the cells into stationary phase. The possibility that the short length of the SSA3 5'-untranslated region is a major determinant of the inefficient translation of SSA3p-UBI4-lacZ in stationary phase is discussed.     

Mitochondrial inheritance in yeast

Mitochondria are the site of oxidative phosphorylation, play a key role in cellular energy metabolism, and are critical for cell survival and proliferation. The propagation of mitochondria during cell division depends on replication and partitioning of mitochondrial DNA, cytoskeleton-dependent mitochondrial transport, intracellular positioning of the organelle, and activities coordinating these processes. Budding yeast Saccharomyces cerevisiae has proven to be a valuable model organism to study the mechanisms that drive segregation of the mitochondrial genome and determine mitochondrial partitioning and behavior in an asymmetrically dividing cell. Here, I review past and recent advances that identified key components and cellular pathways contributing to mitochondrial inheritance in yeast. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.     

Long-term survival during stationary phase: evolution and the GASP phenotype

The traditional view of the stationary phase of the bacterial life cycle, obtained using standard laboratory culture practices, although useful, might not always provide us with the complete picture. Here, the traditional three phases of the bacterial life cycle are expanded to include two additional phases: death phase and long-term stationary phase. In many natural environments, bacteria probably exist in conditions more akin to those of long-term stationary-phase cultures, in which the expression of a wide variety of stress-response genes and alternative metabolic pathways is essential for survival. Furthermore, stressful environments can result in selection for mutants that express the growth advantage in stationary phase (GASP) phenotype.     

CspC and CspD are essential for Caulobacter crescentus stationary phase survival

The cold shock response in bacteria involves the expression of low-molecular weight cold shock proteins (CSPs) containing a nucleic acid-binding cold shock domain (CSD), which are known to destabilize secondary structures on mRNAs, facilitating translation at low temperatures. Caulobacter crescentus cspA and cspB are induced upon cold shock, while cspC and cspD are induced during stationary phase. In this work, we determined a new coding sequence for the cspC gene, revealing that it encodes a protein containing two CSDs. The phenotypes of C. crescentus csp mutants were analyzed, and we found that cspC is important for cells to maintain viability during extended periods in stationary phase. Also, cspC and cspCD strains presented altered morphology, with frequent non-viable filamentous cells, and cspCD also showed a pronounced cell death at late stationary phase. In contrast, the cspAB mutant presented increased viability in this phase, which is accompanied by an altered expression of both cspC and cspD, but the triple cspABD mutant loses this characteristic. Taken together, our results suggest that there is a hierarchy of importance among the csp genes regarding stationary phase viability, which is probably achieved by a fine tune balance of the levels of these proteins.     

Proteasome synthesis and assembly are required for survival during stationary phase

We examined the alterations in 20S proteasome homeostasis, protein oxidation, and cell viability that occur during the stationary phase or chronological model of yeast aging. Data in this report demonstrate that proteasome subunit expression is increased, proteasome composition is altered, and levels of individual proteasome proteolytic activities are elevated during stationary phase-induced aging in Saccharomyces cerevisiae. Despite such alterations, a progressive loss of proteasome-mediated protein degradation and a significant increase in protein oxidation were observed in cells maintained under stationary phase conditions. Deletion of UMP1, a gene necessary for 20S proteasome biogenesis, had no effect on cellular viability under normal growth conditions, but impaired the ability of cells to survive under stationary phase conditions. During stationary phase, the levels of oxidized protein increased more rapidly and to higher levels in the mutant lacking UMP1 than in the wild-type cells. Taken together, these data implicate a role for proteasome synthesis and altered 20S proteasome composition in maintaining viability during stationary phase, and demonstrate that even with these modifications a gradual loss of proteasome-mediated protein degradation occurs during stationary phase-induced aging. These data also suggest a role for impaired proteasome-mediated protein degradation in increased protein oxidation and cell death observed during the aging of eukaryotic cells.     

Aup1p, a yeast mitochondrial protein phosphatase homolog, is required for efficient stationary phase mitophagy and cell survival

Autophagy is a catabolic membrane-trafficking process that occurs in all eukaryotic cells and leads to the hydrolytic degradation of cytosolic material in the vacuolar or lysosomal lumen. Mitophagy, a selective form of autophagy targeting mitochondria, is poorly understood at present. Several recent reports suggest that mitophagy is a selective process that targets damaged mitochondria, whereas other studies imply a role for mitophagy in cell death processes. In a screen for protein phosphatase homologs that functionally interact with the autophagy-dedicated protein kinase Atg1p in yeast, we have identified Aup1p, encoded by Saccharomyces cerevisiae reading frame YCR079w. Aup1p is highly similar to a family of protein phosphatase homologs in animal cells that are predicted to localize to mitochondria based on sequence analysis. Interestingly, we found that Aup1p localizes to the mitochondrial intermembrane space and is required for efficient mitophagy in stationary phase cells. Viability studies demonstrate that Aup1p is required for efficient survival of cells in prolonged stationary phase cultures, implying a pro-survival role for mitophagy under our working conditions. Our data suggest that Aup1p may be part of a signal transduction mechanism that marks mitochondria for sequestration into autophagosomes.     

surA, an Escherichia coli gene essential for survival in stationary phase.

Mutations in genes not required for exponential growth but essential for survival in stationary phase were isolated in an effort to understand the ability of wild-type Escherichia coli cells to remain viable during prolonged periods of nutritional deprivation. The phenotype of these mutations is referred to as Sur- (survival) and the genes are designated sur. The detailed analysis of one of these mutations is presented here. The mutation (surA1) caused by insertion of a mini-Tn10 element defined a new gene located near 1 min on the E. coli chromosome. It was located directly upstream of pdxA and formed part of a complex operon. Evidence is presented supporting the interpretation that cells harboring the surA1 mutation die during stationary phase while similar insertion mutations in other genes of the operon do not lead to a Sur- phenotype. Strains harboring surA1 had a normal doubling time in both rich and minimal medium, but cultures lost viability after several days in stationary phase. Analysis of revertants and suppressors of surA1, which arose after prolonged incubation in stationary phase, indicates that DNA rearrangements (excisions and duplications) occurred in cultures of this strain even when the viable-cell counts were below 10(2) cells per ml. Cells containing suppressing mutations then grew in the same culture to 10(8) cells per ml, taking over the population. The implications of these observations to our understanding of stationary-phase mutagenesis are discussed.     

Mitochondrial inheritance in budding yeast

During the past decade significant advances were made toward understanding the mechanism of mitochondrial inheritance in the yeast Saccharomyces cerevisiae . A combination of genetics, cell-free assays and microscopy has led to the discovery of a great number of components. These fall into three major categories: cytoskeletal elements, mitochondrial membrane components and regulatory proteins. These proteins mediate activities, including movement of mitochondria from mother cells to buds, segregation of mitochondria and mitochondrial DNA, and equal distribution of the organelle between mother cells and buds during yeast cell division.     

Genetic assessment of stationary phase for cells of the yeast Saccharomyces cerevisiae.

Starvation of cells of the yeast Saccharomyces cerevisiae causes cessation of proliferation and acquisition of characteristic physiological properties. The stationary-phase state that results represents a unique developmental state, as shown by a novel conditional phenotype (M. A. Drebot, G. C. Johnston, and R. A. Singer, Proc. Natl. Acad. Sci. USA 84:7948-7952, 1987): mutant cells cannot proliferate at the restrictive temperature when stimulated to reenter the mitotic cell cycle from stationary phase but are unaffected and continue proliferation indefinitely if transferred to the restrictive temperature during exponential growth. We have exploited this reentry mutant phenotype to demonstrate that the same stationary-phase state is generated by nitrogen, sulfur, or carbon starvation and by the cdc25-1 mutation, which conditionally impairs the cyclic AMP-mediated signal transduction pathway. We also show that heat shock, a treatment that elicits physiological perturbations associated with stationary phase, does not cause cells to enter stationary phase. The physiological properties associated with stationary phase therefore do not result from residence in stationary phase but from the stress conditions that bring about stationary phase.     

Cellular radiation effects and hyperthermia: Cytokinetic investigations with stationary phase yeast cells

Summary Wild type diploid yeast, Saccharomyces cerevisiae strain 211, was subjected to 250 kV X-rays or 50° C heat treatment for 30 min or to a combination of both. X-ray exposure took place either in air or in nitrogen. Cell number, percentage of budding cells and cell cycle progression was followed for up to 12 h post irradiation. The distribution of cell cycle stages was determined by flow cytofluorometry. All treatments cause a retardation of cell division rate. Hyperthermia leads mainly to a lengthening of G₁, whereas X-rays arrest the cells reversibly in G₂. The effect of the combined treatment appears to be merely additive. No selective action of hyperthermia on hypoxic cells was found.Dedicated to Prof. Dr. A. Schraub on the occasion of his 70th birthday     

Mitochondrial superoxide and aging: uncoupling-protein activity and superoxide production

Mitochondria are a major source of superoxide, formed by the one-electron reduction of oxygen during electron transport. Superoxide initiates oxidative damage to phospholipids, proteins and nucleic acids. This damage may be a major cause of degenerative disease and aging. In isolated mitochondria, superoxide production on the matrix side of the membrane is particularly high during reversed electron transport to complex I driven by oxidation of succinate or glycerol 3-phosphate. Reversed electron transport and superoxide production from complex I are very sensitive to proton motive force, and can be strongly decreased by mild uncoupling of oxidative phosphorylation. Both matrix superoxide and the lipid peroxidation product 4-hydroxy-trans-2-nonenal can activate uncoupling through endogenous UCPs (uncoupling proteins). We suggest that superoxide releases iron from aconitase, leading to a cascade of lipid peroxidation and the release of molecules such as hydroxy-nonenal that covalently modify and activate the proton conductance of UCPs and other proteins. A function of the UCPs may be to cause mild uncoupling in response to matrix superoxide and other oxidants, leading to lowered proton motive force and decreased superoxide production. This simple feedback loop would constitute a self-limiting cycle to protect against excessive superoxide production, leading to protection against aging, but at the cost of a small elevation of respiration and basal metabolic rate.     

Consequences of cytochrome c oxidase assembly defects for the yeast stationary phase

The assembly of cytochrome c oxidase (COX) is essential for a functional mitochondrial respiratory chain, although the consequences of a loss of assembled COX at yeast stationary phase, an excellent model for terminally differentiated cells in humans, remain largely unexamined. In this study, we show that a wild-type respiratory competent yeast strain at stationary phase is characterized by a decreased oxidative capacity, as seen by a reduction in the amount of assembled COX and by a decrease in protein levels of several COX assembly factors. In contrast, loss of assembled COX results in the decreased abundance of many mitochondrial proteins at stationary phase, which is likely due to decreased membrane potential and changes in mitophagy. In addition to an altered mitochondrial proteome, COX assembly mutants display unexpected changes in markers of cellular oxidative stress at stationary phase. Our results suggest that mitochondria may not be a major source of reactive oxygen species at stationary phase in cells lacking an intact respiratory chain.