Multiple pathways influence mitochondrial inheritance in budding yeast

Yeast mitochondria form a branched tubular network. Mitochondrial inheritance is tightly coupled with bud emergence, ensuring that daughter cells receive mitochondria from mother cells during division. Proteins reported to influence mitochondrial inheritance include the mitochondrial rho (Miro) GTPase Gem1p, Mmr1p, and Ypt11p. A synthetic genetic array (SGA) screen revealed interactions between gem1Delta and deletions of genes that affect mitochondrial function or inheritance, including mmr1Delta. Synthetic sickness of gem1Delta mmr1Delta double mutants correlated with defective mitochondrial inheritance by large buds. Additional studies demonstrated that GEM1, MMR1, and YPT11 each contribute to mitochondrial inheritance. Mitochondrial accumulation in buds caused by overexpression of either Mmr1p or Ypt11p did not depend on Gem1p, indicating these three proteins function independently. Physical linkage of mitochondria with the endoplasmic reticulum (ER) has led to speculation that distribution of these two organelles is coordinated. We show that yeast mitochondrial inheritance is not required for inheritance or spreading of cortical ER in the bud. Moreover, Ypt11p overexpression, but not Mmr1p overexpression, caused ER accumulation in the bud, revealing a potential role for Ypt11p in ER distribution. This study demonstrates that multiple pathways influence mitochondrial inheritance in yeast and that Miro GTPases have conserved roles in mitochondrial distribution.     

Sir2 deletion prevents lifespan extension in 32 long-lived mutants

Activation of Sir2 orthologs is proposed to increase lifespan downstream of dietary restriction. Here, we describe an examination of the effect of 32 different lifespan-extending mutations and four methods of DR on replicative lifespan (RLS) in the short-lived sir2Δ yeast strain. In every case, deletion of SIR2 prevented RLS extension; however, RLS extension was restored when both SIR2 and FOB1 were deleted in several cases, demonstrating that SIR2 is not directly required for RLS extension. These findings indicate that suppression of the sir2Δ lifespan defect is a rare phenotype among longevity interventions and suggest that sir2Δ cells senesce rapidly by a mechanism distinct from that of wild-type cells. They also demonstrate that failure to observe lifespan extension in a short-lived background, such as cells or animals lacking sirtuins, should be interpreted with caution.     

The class V myosin motor protein, Myo2, plays a major role in mitochondrial motility in Saccharomyces cerevisiae

The actin cytoskeleton is essential for polarized, bud-directed movement of cellular membranes in Saccharomyces cerevisiae and thus ensures accurate inheritance of organelles during cell division. Also, mitochondrial distribution and inheritance depend on the actin cytoskeleton, though the precise molecular mechanisms are unknown. Here, we establish the class V myosin motor protein, Myo2, as an important mediator of mitochondrial motility in budding yeast. We found that mutants with abnormal expression levels of Myo2 or its associated light chain, Mlc1, exhibit aberrant mitochondrial morphology and loss of mitochondrial DNA. Specific mutations in the globular tail of Myo2 lead to aggregation of mitochondria in the mother cell. Isolated mitochondria lacking functional Myo2 are severely impaired in their capacity to bind to actin filaments in vitro. Time-resolved fluorescence microscopy revealed a block of bud-directed anterograde mitochondrial movement in cargo binding-defective myo2 mutant cells. We conclude that Myo2 plays an important and direct role for mitochondrial motility and inheritance in budding yeast.     

Complex I is the major site of mitochondrial superoxide production by paraquat

Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride) is widely used as a redox cycler to stimulate superoxide production in organisms, cells, and mitochondria. This superoxide production causes extensive mitochondrial oxidative damage, however, there is considerable uncertainty over the mitochondrial sites of paraquat reduction and superoxide formation. Here we show that in yeast and mammalian mitochondria, superoxide production by paraquat occurs in the mitochondrial matrix, as inferred from manganese superoxide dismutase-sensitive mitochondrial DNA damage, as well as from superoxide assays in isolated mitochondria, which were unaffected by exogenous superoxide dismutase. This paraquat-induced superoxide production in the mitochondrial matrix required a membrane potential that was essential for paraquat uptake into mitochondria. This uptake was of the paraquat dication, not the radical monocation, and was carrier-mediated. Experiments with disrupted mitochondria showed that once in the matrix paraquat was principally reduced by complex I (mammals) or by NADPH dehydrogenases (yeast) to form the paraquat radical cation that then reacted with oxygen to form superoxide. Together this membrane potential-dependent uptake across the mitochondrial inner membrane and the subsequent rapid reduction to the paraquat radical cation explain the toxicity of paraquat to mitochondria.     

Implication of Ca2+ in the regulation of replicative life span of budding yeast

In eukaryotic cells, Ca(2+)-triggered signaling pathways are used to regulate a wide variety of cellular processes. Calcineurin, a highly conserved Ca(2+)/calmodulin-dependent protein phosphatase, plays key roles in the regulation of diverse biological processes in organisms ranging from yeast to humans. We isolated a mutant of the SIR3 gene, implicated in the regulation of life span, as a suppressor of the Ca(2+) sensitivity of zds1Δ cells in the budding yeast Saccharomyces cerevisiae. Therefore, we investigated a relationship between Ca(2+) signaling and life span in yeast. Here we show that Ca(2+) affected the replicative life span (RLS) of yeast. Increased external and intracellular Ca(2+) levels caused a reduction in their RLS. Consistently, the increase in calcineurin activity by either the zds1 deletion or the constitutively activated calcineurin reduced RLS. Indeed, the shortened RLS of zds1Δ cells was suppressed by the calcineurin deletion. Further, the calcineurin deletion per se promoted aging without impairing the gene silencing typically observed in short-lived sir mutants, indicating that calcineurin plays an important role in a regulation of RLS even under normal growth condition. Thus, our results indicate that Ca(2+) homeostasis/Ca(2+) signaling are required to regulate longevity in budding yeast.     

Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae

Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud.     

Yeast replicative life span--the mitochondrial connection

Mitochondria have been associated with aging in many experimental systems through the damaging action of reactive oxygen species. There is more, however, to the connection between mitochondria and Saccharomyces cerevisiae longevity and aging. Induction of the retrograde response, a pathway signaling mitochondrial dysfunction, results in the extension of life span and postponement of the manifestations of aging, changing the metabolic and stress resistance status of the cell. A paradox associated with the retrograde response is the simultaneous triggering of extrachromosomal ribosomal DNA circle (ERC) production, because of the deleterious effect these circles have on yeast longevity. The retrograde response gene RTG2 appears to play a pivotal role in ERC production, linking metabolism and genome stability. In addition to mother cell aging, mitochondria are important in establishment of age asymmetry between mother and daughter cells. The results more generally point to the existence of a mechanism to "filter" damaged components from daughter cells, a form of checkpoint control. Mitochondrial integrity is affected by the PHB1 and PHB2 genes, which encode inner mitochondrial membrane chaperones called prohibitins. The Phb1/2 proteins protect the cell from imbalances in the production of mitochondrial proteins. Such imbalances appear to cause a stochastic stratification of the yeast population with the appearance of short-lived cells. Ras2p impacts this process. Maintenance of mitochondrial membrane potential and the provision of Krebs cycle intermediates for biosyntheses appear to be crucial elements in yeast longevity. In sum, it is clear that mitochondria lie at the nexus of yeast longevity and aging.     

Actin-dependent mitochondrial motility in mitotic yeast and cell-free systems: identification of a motor activity on the mitochondrial surface

Using fluorescent membrane potential sensing dyes to stain budding yeast, mitochondria are resolved as tubular organelles aligned in radial arrays that converge at the bud neck. Time-lapse fluorescence microscopy reveals region-specific, directed mitochondrial movement during polarized yeast cell growth and mitotic cell division. Mitochondria in the central region of the mother cell move linearly towards the bud, traverse the bud neck, and progress towards the bud tip at an average velocity of 49 +/- 21 nm/sec. In contrast, mitochondria in the peripheral region of the mother cell and at the bud tip display significantly less movement. Yeast strains containing temperature sensitive lethal mutations in the actin gene show abnormal mitochondrial distribution. No mitochondrial movement is evident in these mutants after short-term shift to semi-permissive temperatures. Thus, the actin cytoskeleton is important for normal mitochondrial movement during inheritance. To determine the possible role of known myosin genes in yeast mitochondrial motility, we investigated mitochondrial inheritance in myo1, myo2, myo3 and myo4 single mutants and in a myo2, myo4 double mutant. Mitochondrial spatial arrangement and motility are not significantly affected by these mutations. We used a microfilament sliding assay to examine motor activity on isolated yeast mitochondria. Rhodamine-phalloidin labeled yeast actin filaments bind to immobilized yeast mitochondria, as well as unilamellar, right- side-out, sealed mitochondrial outer membrane vesicles. In the presence of low levels of ATP (0.1-100 microM), we observed F-actin sliding on immobilized yeast mitochondria. In the presence of high levels of ATP (500 microM-2 mM), bound filaments are released from mitochondria and mitochondrial outer membranes. The maximum velocity of mitochondria- driven microfilament sliding (23 +/- 11 nm/sec) is similar to that of mitochondrial movement in living cells. This motor activity requires hydrolysis of ATP, does not require cytosolic extracts, is sensitive to protease treatment, and displays an ATP concentration dependence similar to that of members of the myosin family of actin-based motors. This is the first demonstration of an actin-based motor activity in a defined organelle population.     

Live cell imaging of mitochondrial movement along actin cables in budding yeast

BACKGROUND: Mitochondrial inheritance is essential for cell division. In budding yeast, mitochondrial movement from mother to daughter requires (1) actin cables, F-actin bundles that undergo retrograde movement during elongation from buds into mother cells; (2) the mitochore, a mitochondrial protein complex implicated in linking mitochondria to actin cables; and (3) Arp2/3 complex-mediated force generation on mitochondria. RESULTS: We observed three new classes of mitochondrial motility: anterograde movement at velocities of 0.2-0.33 microm/s, retrograde movement at velocities of 0.26-0.51 microm/s, and no net anterograde or retrograde movement. In all cases, motile mitochondria were associated with actin cables undergoing retrograde flow at velocities of 0.18-0.62 microm/s. Destabilization of actin cables or mutations of the mitochore blocked all mitochondrial movements. In contrast, mutations in the Arp2/3 complex affected anterograde but not retrograde mitochondrial movements. CONCLUSIONS: Actin cables are required for movement of mitochondria, secretory vesicles, mRNA, and spindle alignment elements in yeast. We provide the first direct evidence that one of the proposed cargos use actin cables as tracks. In the case of mitochondrial inheritance, anterograde movement drives transfer of the organelle from mothers to buds, while retrograde movement contributes to retention of the organelle in mother cells. Interaction of mitochondria with actin cables is required for anterograde and retrograde movement. In contrast, force generation on mitochondria is required only for anterograde movement. Finally, we propose a novel mechanism in which actin cables serve as "conveyor belts" that drive retrograde organelle movement.     

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.     

Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat

Superoxide is the proximal reactive oxygen species (ROS) produced by the mitochondrial respiratory chain and plays a major role in pathological oxidative stress and redox signaling. While there are tools to detect or decrease mitochondrial superoxide, none can rapidly and specifically increase superoxide production within the mitochondrial matrix. This lack impedes progress, making it challenging to assess accurately the roles of mitochondrial superoxide in cells and in vivo. To address this unmet need, we synthesized and characterized a mitochondria-targeted redox cycler, MitoParaquat (MitoPQ) that comprises a triphenylphosphonium lipophilic cation conjugated to the redox cycler paraquat. MitoPQ accumulates selectively in the mitochondrial matrix driven by the membrane potential. Within the matrix, MitoPQ produces superoxide by redox cycling at the flavin site of complex I, selectively increasing superoxide production within mitochondria. MitoPQ increased mitochondrial superoxide in isolated mitochondria and cells in culture ~a thousand-fold more effectively than untargeted paraquat. MitoPQ was also more toxic than paraquat in the isolated perfused heart and in Drosophila in vivo. MitoPQ enables the selective generation of superoxide within mitochondria and is a useful tool to investigate the many roles of mitochondrial superoxide in pathology and redox signaling in cells and in vivo.     

Mitochondrial and cytosolic expression of human peroxiredoxin 5 in Saccharomyces cerevisiae protect yeast cells from oxidative stress induced by paraquat

Human peroxiredoxin 5 is a recently discovered mitochondrial, peroxisomal and cytosolic thioredoxin peroxidase able to reduce hydrogen peroxide and alkyl hydroperoxides. To gain insight into peroxiredoxin 5 antioxidant role in cell protection, we investigated the resistance of yeast cells expressing human peroxiredoxin 5 in mitochondria or in the cytosol against oxidative stress induced by paraquat. The herbicide paraquat is a redox active drug known to generate superoxide anions in mitochondria and the cytosol of yeast and mammalian cells leading to the formation of several reactive oxygen species. Here, we report that mitochondrial and cytosolic human peroxiredoxin 5 protect yeast cells from cytotoxicity and lipid peroxidation induced by paraquat.     

The role of respiration, reactive oxygen species and oxidative stress in mother cell-specific ageing of yeast strains defective in the RAS signalling pathway

We show that the dominant activated allele of the yeast RAS gene, RAS2(ala18,val19), led to redox imbalance in exponential-phase cells and to excretion of almost all of the cellular glutathione into the medium when the cells reached early-stationary phase. The mitochondria of the mutant stained strongly with dihydrorhodamine 123 (DHR) and the cells displayed a very short mother cell-specific lifespan. Adding 1 mM reduced glutathione (GSH) to the medium partly restored the lifespan. The corresponding RAS2(+) rho-zero strain also displayed a short lifespan, excreted nearly all of its GSH, and stained positively with DHR. Adding 1 mM GSH completely restored the lifespan of the RAS2(+) rho-zero strain to that of the wild-type cells. The double mutant RAS2(ala18,val19) rho-zero cells showed the same lifespan as the RAS2(ala18,val19) cells, and the effect of glutathione in restoring the lifespan was the same, indicating that both mutations shorten lifespan through a similar mechanism. In the RAS2(ala18,val19) mutant strain and its rho-zero derivative we observed for the first time a strong electron spin resonance (ESR) signal characteristic of the superoxide radical anion. The mutant cells were, therefore, producing superoxide in the absence of a complete mitochondrial electron transport chain, pointing to the existence of a possible non-mitochondrial source for ROS generation. Our results indicate that oxidative stress resulting from a disturbance of redox balance can play a major role in mother cell-specific lifespan determination of yeast cells.     

Loss of the mitochondrial lipid cardiolipin leads to decreased glutathione synthesis

Previous studies demonstrated that loss of CL in the yeast mutant crd1Δ leads to perturbation of mitochondrial iron‑sulfur (FeS) cluster biogenesis, resulting in decreased activity of mitochondrial and cytosolic Fe-S-requiring enzymes, including aconitase and sulfite reductase. In the current study, we show that crd1Δ cells exhibit decreased levels of glutamate and cysteine and are deficient in the essential antioxidant, glutathione, a tripeptide of glutamate, cysteine, and glycine. Glutathione is the most abundant non-protein thiol essential for maintaining intracellular redox potential in almost all eukaryotes, including yeast. Consistent with glutathione deficiency, the growth defect of crd1Δ cells at elevated temperature was rescued by supplementation of glutathione or glutamate and cysteine. Sensitivity to the oxidants iron (FeSO₄) and hydrogen peroxide (H₂O₂), was rescued by supplementation of glutathione. The decreased intracellular glutathione concentration in crd1Δ was restored by supplementation of glutamate and cysteine, but not by overexpressing YAP1, an activator of expression of glutathione biosynthetic enzymes. These findings show for the first time that CL plays a critical role in regulating intracellular glutathione metabolism.     

Deletion of the mitochondrial Pim1/Lon protease in yeast results in accelerated aging and impairment of the proteasome

The Saccharomyces cerevisiae homolog of the ATP-dependent Lon protease, Pim1p, is essential for mitochondrial protein quality control, DNA maintenance, and respiration. Here, we demonstrate that Pim1p activity declines in aging cells and that Pim1p deficiency shortens the replicative life span of yeast mother cells. This accelerated aging of pim1Δ cells is accompanied by elevated cytosolic levels of oxidized and aggregated proteins, as well as reduced proteasome activity. Overproduction of Hsp104p greatly diminishes aggregation of oxidized cytosolic proteins, rescues proteasome activity, and restores life span of pim1Δ cells to near wild-type levels. Our results show that defects in mitochondrial protein quality control have global intracellular effects leading to the increased generation of misfolded proteins and cytosolic protein aggregates, which are linked to a decline in replicative potential.     

A fraction of yeast Cu,Zn-superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondria. A physiological role for SOD1 in guarding against mitochondrial oxidative damage

Cu,Zn-superoxide dismutase (SOD1) is an abundant, largely cytosolic enzyme that scavenges superoxide anions. The biological role of SOD1 is somewhat controversial because superoxide is thought to arise largely from the mitochondria where a second SOD (manganese SOD) already resides. Using bakers' yeast as a model, we demonstrate that Cu,Zn-SOD1 helps protect mitochondria from oxidative damage, as sod1Delta mutants show elevated protein carbonyls in this organelle. In accordance with this connection to mitochondria, a fraction of active SOD1 localizes within the intermembrane space (IMS) of mitochondria together with its copper chaperone, CCS. Neither CCS nor SOD1 contains typical N-terminal presequences for mitochondrial uptake; however, the mitochondrial accumulation of SOD1 is strongly influenced by CCS. When CCS synthesis is repressed, mitochondrial SOD1 is of low abundance, and conversely IMS SOD1 is very high when CCS is largely mitochondrial. The mitochondrial form of SOD1 is indeed protective against oxidative damage because yeast cells enriched for IMS SOD1 exhibit prolonged survival in the stationary phase, an established marker of mitochondrial oxidative stress. Cu,Zn-SOD1 in the mitochondria appears important for reactive oxygen physiology and may have critical implications for SOD1 mutations linked to the fatal neurodegenerative disorder, amyotrophic lateral sclerosis.     

Yeast mitochondria contain ATP-sensitive, reversible actin-binding activity.

Sedimentation assays were used to demonstrate and characterize binding of isolated yeast mitochondria to phalloidin-stabilized yeast F-actin. These actin-mitochondrial interactions are ATP sensitive, saturable, reversible, and do not depend upon mitochondrial membrane potential. Protease digestion of mitochondrial outer membrane proteins or saturation of myosin-binding sites on F-actin with the S1 subfragment of skeletal myosin block binding. These observations indicate that a protein (or proteins) on the mitochondrial surface mediates ATP-sensitive, reversible binding of mitochondria to the lateral surface of microfilaments. Actin copurifies with mitochondria during subcellular fractionation and is released from the organelle upon treatment with ATP. Thus, actin-mitochondrial interactions resembling those observed in vitro may also exist in intact yeast cells. Finally, a yeast mutant bearing a temperature-sensitive mutation in the actin-encoding ACT1 gene (act1-3) displays temperature-dependent defects in transfer of mitochondria from mother cells to newly developed buds during yeast cell mitosis.     

A type V myosin (Myo2p) and a Rab-like G-protein (Ypt11p) are required for retention of newly inherited mitochondria in yeast cells during cell division

Two actin-dependent force generators contribute to mitochondrial inheritance: Arp2/3 complex and the myosin V Myo2p (together with its Rab-like binding partner Ypt11p). We found that deletion of YPT11, reduction of the length of the Myo2p lever arm (myo2-Delta6IQ), or deletion of MYO4 (the other yeast myosin V), had no effect on mitochondrial morphology, colocalization of mitochondria with actin cables, or the velocity of bud-directed mitochondrial movement. In contrast, retention of mitochondria in the bud was compromised in YPT11 and MYO2 mutants. Retention of mitochondria in the bud tip of wild-type cells results in a 60% decrease in mitochondrial movement in buds compared with mother cells. In ypt11Delta mutants, however, the level of mitochondrial motility in buds was similar to that observed in mother cells. Moreover, the myo2-66 mutant, which carries a temperature-sensitive mutation in the Myo2p motor domain, exhibited a 55% decrease in accumulation of mitochondria in the bud tip, and an increase in accumulation of mitochondria at the retention site in the mother cell after shift to restrictive temperatures. Finally, destabilization of actin cables and the resulting delocalization of Myo2p from the bud tip had no significant effect on the accumulation of mitochondria in the bud tip.     

Yeast mitochondrial division and distribution require the cortical num1 protein

Yeast mitochondrial division requires the dynamin-related Dnm1 protein. By isolating high-copy suppressors of a dominant-negative Dnm1p mutant, we uncovered an unexpected role in mitochondrial division and inheritance for Num1p, a protein previously shown to facilitate nuclear migration. num1 mutants contain an interconnected network of mitochondrial tubules, remarkably similar to cells lacking Dnm1p, and time-lapse microscopy confirms that mitochondrial fission is greatly reduced in num1Delta cells. We also find that Num1p assembles into punctate structures, which often colocalize with mitochondrial-bound Dnm1p particles. Suggesting a role for both Num1p and Dnm1p in mitochondrial inheritance, we find that num1 dnm1 double mutants accumulate mitochondria in daughter buds and that mother cells are frequently devoid of all mitochondria. Thus, our studies have revealed an additional role for Dnm1p in mitochondrial transmission through its interaction with Num1p, thereby providing a link between mitochondrial division and inheritance.