Mitochondrial quality control during inheritance is associated with lifespan and mother-daughter age asymmetry in budding yeast

Fluorescence loss in photobleaching experiments and analysis of mitochondrial function using superoxide and redox potential biosensors revealed that mitochondria within individual yeast cells are physically and functionally distinct. Mitochondria that are retained in mother cells during yeast cell division have a significantly more oxidizing redox potential and higher superoxide levels compared to mitochondria in buds. Retention of mitochondria with more oxidizing redox potential in mother cells occurs to the same extent in young and older cells and can account for the age-associated decline in total cellular mitochondrial redox potential in yeast as they age from 0 to 5 generations. Deletion of Mmr1p, a member of the DSL1 family of tethering proteins that localizes to mitochondria at the bud tip and is required for normal mitochondrial inheritance, produces defects in mitochondrial quality control and heterogeneity in replicative lifespan (RLS). Long-lived mmr1Δ cells exhibit prolonged RLS, reduced mean generation times, more reducing mitochondrial redox potential and lower mitochondrial superoxide levels compared to wild-type cells. Short-lived mmr1Δ cells exhibit the opposite phenotypes. Moreover, short-lived cells give rise exclusively to short-lived cells, while the majority of daughters of long-lived cells are long lived. These findings support the model that the mitochondrial inheritance machinery promotes retention of lower-functioning mitochondria in mother cells and that this process contributes to both mother-daughter age asymmetry and age-associated declines in cellular fitness.     

A role for ubiquitination in mitochondrial inheritance in Saccharomyces cerevisiae.

The smm1 mutation suppresses defects in mitochondrial distribution and morphology caused by the mdm1-252 mutation in the yeast Saccharomyces cerevisiae. Cells harboring only the smm1 mutation themselves display temperature-sensitive growth and aberrant mitochondrial inheritance and morphology at the nonpermissive temperature. smm1 maps to RSP5, a gene encoding an essential ubiquitin-protein ligase. The smm1 defects are suppressed by overexpression of wild-type ubiquitin but not by overexpression of mutant ubiquitin in which lysine-63 is replaced by arginine. Furthermore, overexpression of this mutant ubiquitin perturbs mitochondrial distribution and morphology in wild-type cells. Site-directed mutagenesis revealed that the ubiquitin ligase activity of Rsp5p is essential for its function in mitochondrial inheritance. A second mutation, smm2, which also suppressed mdm1-252 defects, but did not cause aberrant mitochondrial distribution and morphology, mapped to BUL1, encoding a protein interacting with Rsp5p. These results indicate that protein ubiquitination mediated by Rsp5p plays an essential role in mitochondrial inheritance, and reveal a novel function for protein ubiquitination.     

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.     

INVITED SPECIAL PAPER: The hows and whys of cytoplasmic inheritance in seed plants

Cytoplasmic organelles are inherited in a nonMendelian fashion in all eukaryotic organisms investigated. Among the seed plants, plastids can be inherited strictly from the female parent, strictly from the male parent, or biparentally. Most flowering plants studied to date exhibit maternal plastid inheritance, but approximately one-third of the genera investigated display biparental plastid inheritance to some degree. Among the gymnosperms, paternal plastid inheritance is the rule in the conifers, whereas the other groups appear to have maternal plastid inheritance, although they have been less well studied. Mitochondrial inheritance is predominantly maternal in the seed plants, except for a few coniferous families where it is predominantly paternal. The advent of recombinant DNA technology has allowed restriction fragment length polymorphisms to be used as molecular markers, and has stimulated much research in organelle inheritance and its application to studies of population genetics and phylogenetic biology. This report emphasizes the various mechanisms by which organelles are, or are not, transmitted among the seed plants in order that researchers directly or indirectly involved with organelle inheritance may better understand the potential and the limitations of their investigations. A summary and discussion of the possible evolutionary significance of the various patterns of cytoplasmic inheritance among the seed plants are also included.     

Organelle inheritance in plant cell division: the actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts

Nuclear inheritance is highly ordered, ensuring stringent, unbiased partitioning of chromosomes before cell division. In plants, however, little is known about the analogous cellular processes that might ensure unbiased inheritance of non-nuclear organelles, either in meristematic cell divisions or those induced during the acquisition of totipotency. We have investigated organelle redistribution and inheritance mechanisms during cell division in cultured tobacco mesophyll protoplasts. Quantitative analysis of organelle repositioning observed by autofluorescence of chloroplasts or green fluorescent protein (GFP), targeted to mitochondria or endoplasmic reticulum (ER), demonstrated that these organelles redistribute in an ordered manner before division. Treating protoplasts with cytoskeleton-disrupting drugs showed that redistribution depended on actin filaments (AFs), but not on microtubules (MTs), and furthermore, that an intact actin cytoskeleton was required to achieve unbiased organelle inheritance. Labelling the actin cytoskeleton with a novel GFP-fusion protein revealed a highly dynamic actin network, with local reorganisation of this network itself, appearing to contribute substantially to repositioning of chloroplasts and mitochondria. Our observations show that each organelle exploits a different strategy of redistribution to ensure unbiased partitioning. We conclude that inheritance of chloroplasts, mitochondria and ER in totipotent plant cells is an ordered process, requiring complex interactions with the actin cytoskeleton.     

The yeast dynamin-like protein, Mgm1p, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance

The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37 degrees C, while loss of the mitochondrial genome occurred after 4-24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1-null mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.     

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.     

Mitochondrial dynamics and division in budding yeast

Mitochondria adopt a variety of different shapes in eukaryotic cells, ranging from multiple, small compartments to elaborate tubular networks. The establishment and maintenance of different mitochondrial morphologies depends, in part, on the equilibrium between opposing fission and fusion events. Recent studies in yeast, flies, worms and mammalian cells indicate that three high-molecular-weight GTPases control mitochondrial membrane dynamics. One of these is a dynamin-related GTPase that acts on the outer mitochondrial membrane to regulate fission. Recently, genetic approaches in budding yeast have identified additional components of the fission machinery. These and other new findings suggest a common mechanism for membrane fission events that has been conserved and adapted during eukaryotic evolution.     

Cell polarization and cytokinesis in budding yeast

Asymmetric cell division, which includes cell polarization and cytokinesis, is essential for generating cell diversity during development. The budding yeast Saccharomyces cerevisiae reproduces by asymmetric cell division, and has thus served as an attractive model for unraveling the general principles of eukaryotic cell polarization and cytokinesis. Polarity development requires G-protein signaling, cytoskeletal polarization, and exocytosis, whereas cytokinesis requires concerted actions of a contractile actomyosin ring and targeted membrane deposition. In this chapter, we discuss the mechanics and spatial control of polarity development and cytokinesis, emphasizing the key concepts, mechanisms, and emerging questions in the field.     

Mitochondrial DNA and RNA isolation from small amounts of potato tissue

We present a fast and simple protocol for purification of mitochondrial DNA and RNA from small amounts of potato tissue including tubers, leaves, flowers, and flower buds. This method uses a high ionic strength medium to isolate mitochondria and extract mitochondrial DNA and RNA from a single preparation and is easily adaptable to other plant species. The mitochondrial DNA was not contaminated by plastid DNA, was fully restrictable and was successfully used for PCR, cloning and Southern analyses. Similarly, the isolated mitochondrial RNA was not contaminated (flower buds) or only slightly contaminated (leaves) by plastid RNA. RNA prepared according to our method was acceptable for northern and RT-PCR analyses.     

Sharing the wealth: peroxisome inheritance in budding yeast

Eukaryotic cells have evolved molecular mechanisms to ensure the faithful partitioning of cellular components during cell division. The budding yeast Saccharomyces cerevisiae has to actively deliver about half of its organelles to the growing bud, while retaining the remaining organelles in the mother cell. Until lately, little was known about the inheritance of peroxisomes. Recent studies have identified the peroxisomal proteins Inp1p and Inp2p as two key regulators of peroxisome inheritance that perform antagonistic functions. Inp1p is required for the retention of peroxisomes in mother cells, whereas Inp2p promotes the bud-directed movement of these organelles. Inp1p anchors peroxisomes to the cell cortex by interacting with specific structures lining the cell periphery. On the other hand, Inp2p functions as the peroxisome-specific receptor for the class V myosin, Myo2p, thereby linking peroxisomes to the translocation machinery that propels peroxisome movement. Tight coordination between Inp1p and Inp2p ensures a fair and harmonious spatial segregation of peroxisomes upon cell division.     

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.     

An ER‐peroxisome tether exerts peroxisome population control in yeast

Eukaryotic cells compartmentalize biochemical reactions into membrane‐enclosed organelles that must be faithfully propagated from one cell generation to the next. Transport and retention processes balance the partitioning of organelles between mother and daughter cells. Here we report the identification of an ER‐peroxisome tether that links peroxisomes to the ER and ensures peroxisome population control in the yeast Saccharomyces cerevisiae. The tether consists of the peroxisome biogenic protein, Pex3p, and the peroxisome inheritance factor, Inp1p. Inp1p bridges the two compartments by acting as a molecular hinge between ER‐bound Pex3p and peroxisomal Pex3p. Asymmetric peroxisome division leads to the formation of Inp1p‐containing anchored peroxisomes and Inp1p‐deficient mobile peroxisomes that segregate to the bud. While peroxisomes in mother cells are not released from tethering, de novo formation of tethers in the bud assists in the directionality of peroxisome transfer. Peroxisomes are thus stably maintained over generations of cells through their continued interaction with tethers.     

Genetically encoded multimode reporter of adaptor complex 3 traffic in budding yeast

AP‐3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. In mammals, mutations in AP‐3 cause Hermansky‐Pudlak syndrome type 2, cyclic neutropenias, and a form of epileptic encephalopathy. In budding yeast, AP‐3 carries cargo directly from the trans‐Golgi to the lysosomal vacuole. Despite the pathway's importance and its discovery two decades ago, rapid screens and selections for AP‐3 mutants have not been available. We now report GNSI, a synthetic, genetically encoded reporter that allows rapid plate‐based assessment of AP‐3 functional deficiency, using either chromogenic or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP‐3 carrier vesicles and is adaptable to high‐throughput screening or selection in both plate array and liquid batch culture formats. Episomal and integrating plasmids encoding GNSI have been submitted to the Addgene repository.     

Vac8p, an armadillo repeat protein, coordinates vacuole inheritance with multiple vacuolar processes

Vac8p, an armadillo (ARM) repeat protein, is required for multiple vacuolar processes. It functions in vacuole inheritance, cytoplasm-to-vacuole protein targeting pathway, formation of the nucleus-vacuole junction and vacuole-vacuole fusion. These functions each utilize a distinct Vac8p-binding partner. Here, we report an additional Vac8p function: caffeine resistance. We show that Vac8p function in caffeine resistance is mediated via a newly identified Vac8p-binding partner, Tco89p. The interaction between Vac8p and each binding partner requires an overlapping subset of Vac8p ARM repeats. Moreover, these partners can compete with each other for access to Vac8p. Furthermore, Vac8p is enriched in three separate subdomains on the vacuole, each with a unique binding partner dedicated to a different vacuolar function. These findings suggest that a major role of Vac8p is to spatially separate multiple functions thereby enabling vacuole inheritance to occur concurrently with other vacuolar processes.     

Direct evidence for the interaction of platelet surface membrane proteins GPIIb and III with cytoskeletal components: protein crosslinking studies

When intact platelets are incubated at 37 degrees C with Concanavalin A (ConA), the two major surface membrane proteins GPIIb and III become associated with the Triton-insoluble cytoskeleton. Preincubation of platelets with a variety of metabolic inhibitors, including cytochalasin B, 2-deoxy-D-glucose, and antimycin A or lidocaine, had no effect on the ability of ConA to produce this effect. These results suggested that the ConA-induced anchorage of GPIIb and III to the Triton-insoluble cytoskeleton is a passive process requiring clustering of GPIIb-III molecules but not requiring the metabolic energy of an intact cell. This was supported by experiments that showed that ConA binding to plasma membrane-rich fractions at 37 degrees C could induce association of GPIIb and III with a sedimentable actin-rich, Triton-insoluble membrane matrix. Similar results were obtained when membranes were first isolated from ConA-treated cells. Adding DNAse I, an actin depolymerizing agent, into the Triton extraction buffer inhibited the ConA-induced sedimentation of GPIIb-III and actin by 50% in the presence of Mg2+-ATP. Treatment of ConA-treated membranes with dimethyl-3,3'-dithiobispropiomidate, a bifunctional, reducible protein crosslinking agent, produced Triton-insoluble crosslinked species of discrete molecular weights. When these cross-linked species were analyzed by SDS-PAGE in the presence of beta-mercaptoethanol, they were found to be composed of a 180-200 K dalton protein, GPIIb, GPIII, and actin. Crosslinking of these components was equally effective after Triton treatment and indicated as well that the species crosslinked in the intact membrane was stable after Triton extraction. Addition of crosslinker to membranes not treated with ConA produced similar crosslinked species. Analysis of their composition on reduced gels revealed that the amounts of GPIIb and III were reduced greatly (less than 10% of the total input GPIIb and III) but that the 180-200 k dalton protein and actin content were similar to that seen with ConA-treated membranes. These results are consistent with the notion that ConA clusters mobile, unanchored molecules of GPIIb-III (approximately 90-95% of the total) around a small fraction of IIb-III that is associated with a submembranous cytoskeleton.     

In Vivo Evidence That the Stromelysin-3 Metalloproteinase Contributes in a Paracrine Manner to Epithelial Cell Malignancy

Stromelysin-3 (ST3; Basset, P., J.P. Bellocq, C. Wolf, I. Stoll, P. Hutin, J.M. Limacher, O.L. Podhajcer, M.P. Chenard, M.C. Rio, P. Chambon. 1990. Nature. 348:699–704) is a matrix metalloproteinase (MMP) expressed in mesenchymal cells located close to epithelial cells, during physiological and pathological tissue remodeling processes. In human carcinomas, high ST3 levels are associated with a poor clinical outcome, suggesting that ST3 plays a role during malignant processes. In this study we report the ST3 gene inactivation by homologous recombination. Although ST3 null mice (ST3^−/−) were fertile and did not exhibit obvious alterations in appearance and behavior, the lack of ST3 altered malignant processes. Thus, the suppression of ST3 results in a decreased 7,12-dimethylbenzanthracene-induced tumorigenesis in ST3^−/− mice. Moreover, ST3^−/− fibroblasts have lost the capacity to promote implantation of MCF7 human malignant epithelial cells in nude mice (P < 0.008). Finally, we show that this ST3 paracrine function requires extracellular matrix (ECM)-associated growth factors. Altogether, these findings give evidence that ST3 promotes, in a paracrine manner, homing of malignant epithelial cells, a key process for both primary tumors and metastases. Therefore, ST3 represents an appropriate target for specific MMP inhibitor(s) in future therapeutical approaches directed against the stromal compartment of human carcinomas.