Control of mitochondrial volume by mitochondrial metabolic water.

It is well-known that mitochondrial volume largely controls mitochondrial functioning. We investigate whether metabolic water produced by oxidative phosphorylation could be involved in mitochondrial volume regulation. We modulated the generation of this water in liver mitochondria and assess their volume by two independent techniques. In liver mitochondria, the mitochondrial volume was specifically decreased when no water was produced independently of energetic parameters and uncoupling activity. In all other conditions associated with water generation, there was no significant change in mitochondrial metabolic volume. Altogether these data demonstrate that mitochondrial volume is regulated, independently of energetic status, by the mitochondrial metabolic water that acts as a signal.

Selective and non-selective autophagic degradation of mitochondria in yeast.

Mitochondria are essential to oxidative energy production in aerobic eukaryotic cells, where they are also required for multiple biosynthetic pathways to take place. Mitochondrial homeostasis also plays a crucial role in ageing and programmed cell death, and recent data have suggested that mitochondria degradation is a strictly regulated process. Autophagy is an evolutionary conserved mechanism that provides cells with a mechanism for the continuous turnover of damaged and obsolete macromolecules and organelles. In this work, we investigated mitochondria degradation by autophagy. Electron microscopy observations of yeast cells submitted to nitrogen starvation after growth on different carbon sources provided evidence that microautophagy, rather than macroautophagy, preferentially occurred in cells grown under nonfermentable conditions. The observation of mitochondria degradation showed that both a selective process and a nonselective process of mitochondria autophagy occurred successively. In a yeast strain inactivated for the gene UTH1, the selective process was not observed.

Evaluation of the roles of apoptosis, autophagy, and mitophagy in the loss of plating efficiency induced by Bax expression in yeast.

We found recently that, in yeast cells, the heterologous expression of Bax induces a loss of plating efficiency different from that induced by acute stress because it is associated with the maintenance of plasma membrane integrity (Camougrand, N., Grelaud-Coq, A., Marza, E., Priault, M., Bessoule, J. J., and Manon, S. (2003) Mol. Microbiol. 47, 495-506). Bax effects were neither dependent on the presence of the yeast metacaspase Yca1p and the apoptosis-inducing factor homolog nor associated with the appearance of typical apoptotic markers such as metacaspase activation, annexin V binding, and DNA cleavage. Yeast cells expressing Bax instead displayed autophagic features, including increased accumulation of Atg8p, activation of vacuolar alkaline phosphatase, and the presence of autophagosomes and autophagic bodies. However, the inactivation of autophagy did not prevent and actually slightly accelerated Bax-induced loss of plating efficiency. On the other hand, Bax expression induced a fragmentation of the mitochondrial network, which retained, however, some level of organization in wild-type cells. However, when expressed in cells inactivated for the gene UTH1, previously shown to be involved in mitophagy, Bax induced a complete disorganization of the mitochondrial network. Interestingly, although mitochondrially targeted green fluorescent protein was slowly degraded in the wild-type strain, it remained unaffected in the mutant. Furthermore, the slow loss of plating efficiency in the mutant strain correlated with a loss of plasma membrane integrity. These data suggest that Bax-induced loss of growth capacity is associated with maintenance of plasma membrane integrity dependent on UTH1, suggesting that selective degradation of altered mitochondria is required for a regulated loss of growth capacity.

Transcriptome analysis of bud burst in sessile oak (Quercus petraea).

Expression patterns of hundreds of transcripts in apical buds were monitored during bud flushing in sessile oak (Quercus petraea), in order to identify genes differentially expressed between the quiescent and active stage of bud development. Different transcriptomic techniques combining the construction of suppression subtractive hybridization (SSH) libraries and the monitoring of gene expression using macroarray and real-time reverse transcriptase polymerase chain reaction (RT-PCR) were performed to dissect bud burst, with a special emphasis on the onset of the process. We generated 801 expressed sequence tags (ESTs) derived from six developmental stages of bud burst. Macroarray experiment revealed a total of 233 unique transcripts exhibiting differential expression during the process, and a putative function was assigned to 65% of them. Cell rescue/defense-, metabolism-, protein synthesis-, cell cycle- and transcription-related transcripts were among the most regulated genes. Macroarray and real-time RT-PCR showed that several genes exhibited contrasted expressions between quiescent and swelling buds, such as a putative homologue of the transcription factor DAG2 (Dof Affecting Germination 2), previously reported to be involved in the control of seed germination in Arabidopsis thaliana. These differentially expressed genes constitute relevant candidates for signaling pathway of bud burst in trees.

Site specific alterations of adipose tissue mitochondria in 3'-azido-3'-deoxythymidine (AZT)-treated rats: an early stage in lipodystrophy?

Although it is well accepted that treatment with nucleoside reverse transcriptase inhibitors (NRTIs) modifies fat metabolism and fat distribution in humans, the mechanisms underlying these modifications are not yet known. The present investigation examines the effects of chronic oral administration of 3'-azido-3'-deoxythymidine (AZT) on the mitochondrial metabolism and the redox status management of rat white adipose tissues originating from two anatomical sites, as well as of the rat liver. Results showed that AZT treatment induced differential effects on the mitochondrial functions depending on the anatomical localisation. Indeed, in inguinal adipose tissue, a significant decrease in the cytochrome c oxidase activity and in the mitochondrial DNA (mtDNA) content was observed, whereas the activity of citrate synthase, a mitochondrial protein exclusively encoded by the nucleus, was not affected. In contrast, no significant change in these parameters could be detected for epididymal tissue and for liver. In parallel, no oxidative stress could be detected after treatment, for both white adipose tissues and for liver, even though treated liver exhibited several modifications in redox management. Taken together, these data demonstrate differential mitochondrial effects of AZT on subcutaneous versus visceral white adipose tissue. Moreover, the decrease in mitochondrial oxidative capacity of inguinal adipocyte consecutive to AZT treatment is not primarily due to an oxidative stress per se, but rather to a depletion of the mtDNA content per cell.

Guanylic nucleotide starvation affects Saccharomyces cerevisiae mother-daughter separation and may be a signal for entry into quiescence.

BACKGROUND: Guanylic nucleotides are both macromolecules constituents and crucial regulators for a variety of cellular processes. Therefore, their intracellular concentration must be strictly controlled. Consistently both yeast and mammalian cells tightly correlate the transcription of genes encoding enzymes critical for guanylic nucleotides biosynthesis with the proliferation state of the cell population. RESULTS: To gain insight into the molecular relationships connecting intracellular guanylic nucleotide levels and cellular proliferation, we have studied the consequences of guanylic nucleotide limitation on Saccharomyces cerevisiae cell cycle progression. We first utilized mycophenolic acid, an immunosuppressive drug that specifically inhibits inosine monophosphate dehydrogenase, the enzyme catalyzing the first committed step in de novo GMP biosynthesis. To approach this system physiologically, we next developed yeast mutants for which the intracellular guanylic nucleotide pools can be modulated through changes of growth conditions. In both the pharmacological and genetic approaches, we found that guanylic nucleotide limitation generated a mother-daughter separation defect, characterized by cells with two unseparated daughters. We then showed that this separation defect resulted from cell wall perturbations but not from impaired cytokinesis. Importantly, cells with similar separation defects were found in a wild type untreated yeast population entering quiescence upon nutrient limitation. CONCLUSION: Our results demonstrate that guanylic nucleotide limitation slows budding yeast cell cycle progression, with a severe pause in telophase. At the cellular level, guanylic nucleotide limitation causes the emergence of cells with two unseparated daughters. By fluorescence and electron microscopy, we demonstrate that this phenotype arises from defects in cell wall partition between mother and daughter cells. Because cells with two unseparated daughters are also observed in a wild type population entering quiescence, our results reinforce the hypothesis that guanylic nucleotide intracellular pools contribute to a signal regulating both cell proliferation and entry into quiescence.

Failure to assemble the alpha 3 beta 3 subcomplex of the ATP synthase leads to accumulation of the alpha and beta subunits within inclusion bodies and the loss of mitochondrial cristae in Saccharomyces cerevisiae.

The F(1) component of mitochondrial ATP synthase is an oligomeric assembly of five different subunits, alpha, beta, gamma, delta, and epsilon. In terms of mass, the bulk of the structure ( approximately 90%) is provided by the alpha and beta subunits, which form an (alphabeta)(3) hexamer with adenine nucleotide binding sites at the alpha/beta interfaces. We report here ultrastructural and immunocytochemical analyses of yeast mutants that are unable to form the alpha(3)beta(3) oligomer, either because the alpha or the beta subunit is missing or because the cells are deficient for proteins that mediate F assembly (e.g. Atp11p, Atp12p, or Fmc1p). The F(1) alpha(1) and beta subunits of such mutant strains are detected within large electron-dense particles in the mitochondrial matrix. The composition of the aggregated species is principally full-length F(1) alpha and/or beta subunit protein that has been processed to remove the amino-terminal targeting peptide. To our knowledge this is the first demonstration of mitochondrial inclusion bodies that are formed largely of one particular protein species. We also show that yeast mutants lacking the alpha(3)beta(3) oligomer are devoid of mitochondrial cristae and are severely deficient for respiratory complexes III and IV. These observations are in accord with other studies in the literature that have pointed to a central role for the ATP synthase in biogenesis of the mitochondrial inner membrane.

The modulation in subunits e and g amounts of yeast ATP synthase modifies mitochondrial cristae morphology.

Subunits e and g of Saccharomyces cerevisiae ATP synthase are required to maintain ATP synthase dimeric forms. Mutants devoid of these subunits display anomalous mitochondrial morphologies. An expression system regulated by doxycycline was used to modulate the expression of the genes encoding the subunits e and g. A decrease in the amount of subunit e induces a decrease in the amount of subunit g, but a decrease in the amount of subunit g does not affect subunit e. The loss of subunit e or g leads to the loss of supramolecular structures of ATP synthase, which is fully reversible upon removal of doxycycline. In the absence of doxycycline, mitochondria present poorly defined cristae. In the presence of doxycycline, onion-like structures are formed after five generations. When doxycycline is removed after five generations, cristae are mainly observed. The data demonstrate that the inner structure of mitochondria depends upon the ability of ATP synthase to make supramolecular structures.

Isolation and properties of promitochondria from anaerobic stationary-phase yeast cells.

Under anaerobiosis, the mitochondrion of Saccharomyces cerevisiae is restricted to unstructured promitochondria. These promitochondria provide unknown metabolic functions that are required for growth. Since high glucose concentrations are mainly fermented by S. cerevisiae during stationary phase (due to nitrogen starvation), an optimized promitochondria isolation procedure was investigated. Firstly, the unusual promitochondria ultrastructure was checked in intact cells by electron microscopy using a cryo-fixation and freeze-substitution method. The rapid response of anaerobic cells toward oxygen justified the adoption of several critical steps, especially during spheroplasting. Control of spheroplasting was accompanied by a systematic analysis of spheroplast integrity, which greatly influence the final quality of promitochondria. Despite the presence of remnant respiratory chain components under anaerobiosis, characterization of isolated promitochondria by high-resolution respirometry did not reveal any antimycin A- and myxothiazol-sensitive NADH and NADPH oxidase activities. Moreover, the existence of a cyanide-sensitive and non-phosphorylating NADH-dependent oxygen consumption in promitochondria was demonstrated. Nevertheless, promitochondria only slightly contribute to the overall oxygen consumption capacity observed in highly glucose-repressed anaerobic cells.

Regional differences in oxidative capacity of rat white adipose tissue are linked to the mitochondrial content of mature adipocytes.

Two metabolic pathways of the white adipocytes (i.e. de novo lipogenesis and lipolysis) require mitochondria functionality. In this report, the oxidative capacity of two white adipose tissues of rat and their respective isolated adipocytes were evaluated. Two major white fat pads, namely inguinal and epididymal tissues, were chosen as subcutaneous and visceral adipose tissues, respectively. The mitochondrial content of these tissues was estimated using cytological and biochemical analysis. Electron microscopy analysis showed higher mitochondrial density in epididymal than in inguinal adipocytes. The mitochondrial DNA content and mitochondrial enzymatic equipment were also higher in the former than in the latter tissue. A positive correlation between two mitochondrial enzymatic activities, namely cytochrome c oxidase and citrate synthase, and the mtDNA content of adipose tissue was reported. Moreover, NRF1 protein, which belongs to the transcriptional activator family and is thought to be involved in mitochondrial biogenesis regulation, was present in higher proportions in nuclei isolated from epididymal cells than in those from inguinal cells. Finally, greater abundance of mitochondria in epididymal tissue is in agreement with higher cytochrome c oxidase activity as well as increased respiration (i.e. basal and noradrenaline-stimulated) of adipocytes isolated from epididymal tissue as compared to adipocytes isolated from inguinal tissue. Therefore, white adipose tissue appears as a heterogeneous organ with marked variation in mitochondrial content depending on its anatomical location.

The GxxxG motif of the transmembrane domain of subunit e is involved in the dimerization/oligomerization of the yeast ATP synthase complex in the mitochondrial membrane.

A conserved putative dimerization GxxxG motif located in the unique membrane-spanning segment of the ATP synthase subunit e was altered in yeast both by insertion of an alanine residue and by replacement of glycine by leucine residues. These alterations led to the loss of subunit g and the loss of dimeric and oligomeric forms of the yeast ATP synthase. Furthermore, as in null mutants devoid of either subunit e or subunit g, mitochondria displayed anomalous morphologies with onion-like structures. By taking advantage of the presence of the endogenous cysteine 28 residue in the wild-type subunit e, disulfide bond formation between subunits e in intact mitochondria was found to increase the stability of an oligomeric structure of the ATP synthase in digitonin extracts. The data show the involvement of the dimerization motif of subunit e in the formation of supramolecular structures of mitochondrial ATP synthases and are in favour of the existence in the inner mitochondrial membrane of associations of ATP synthases whose masses are higher than those of ATP synthase dimers.

Is there a relationship between the supramolecular organization of the mitochondrial ATP synthase and the formation of cristae?

Blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses of detergent mitochondrial extracts have provided evidence that the yeast ATP synthase could form dimers. Cross-linking experiments performed on a modified version of the i-subunit of this enzyme indicate the existence of such ATP synthase dimers in the yeast inner mitochondrial membrane. We also show that the first transmembrane segment of the eukaryotic b-subunit (bTM1), like the two supernumerary subunits e and g, is required for dimerization/oligomerization of ATP synthases. Unlike mitochondria of wild-type cells that display a well-developed cristae network, mitochondria of yeast cells devoid of subunits e, g, or bTM1 present morphological alterations with an abnormal proliferation of the inner mitochondrial membrane. From these observations, we postulate that an anomalous organization of the inner mitochondrial membrane occurs due to the absence of ATP synthase dimers/oligomers. We provide a model in which the mitochondrial ATP synthase is a key element in cristae morphogenesis.

The ATP synthase is involved in generating mitochondrial cristae morphology.

The inner membrane of the mitochondrion folds inwards, forming the cristae. This folding allows a greater amount of membrane to be packed into the mitochondrion. The data in this study demonstrate that subunits e and g of the mitochondrial ATP synthase are involved in generating mitochondrial cristae morphology. These two subunits are non-essential components of ATP synthase and are required for the dimerization and oligomerization of ATP synthase. Mitochondria of yeast cells deficient in either subunits e or g were found to have numerous digitations and onion-like structures that correspond to an uncontrolled biogenesis and/or folding of the inner mitochondrial membrane. The present data show that there is a link between dimerization of the mitochondrial ATP synthase and cristae morphology. A model is proposed of the assembly of ATP synthase dimers, taking into account the oligomerization of the yeast enzyme and earlier data on the ultrastructure of mitochondrial cristae, which suggests that the association of ATP synthase dimers is involved in the control of the biogenesis of the inner mitochondrial membrane.

In the absence of the first membrane-spanning segment of subunit 4(b), the yeast ATP synthase is functional but does not dimerize or oligomerize.

The N-terminal portion of the mitochondrial b-subunit is anchored in the inner mitochondrial membrane by two hydrophobic segments. We investigated the role of the first membrane-spanning segment, which is absent in prokaryotic and chloroplastic enzymes. In the absence of the first membrane-spanning segment of the yeast subunit (subunit 4), a strong decrease in the amount of subunit g was found. The mutant ATP synthase did not dimerize or oligomerize, and mutant cells displayed anomalous mitochondrial morphologies with onion-like structures. This phenotype is similar to that of the null mutant in the ATP20 gene that encodes subunit g, a component involved in the dimerization/oligomerization of ATP synthase. Our data indicate that the first membrane-spanning segment of the mitochondrial b-subunit is not essential for the function of the enzyme since its removal did not directly alter the oxidative phosphorylation. It is proposed that the unique membrane-spanning segment of subunit g and the first membrane-spanning segment of subunit 4 interact, as shown by cross-linking experiments. We hypothesize that in eukaryotic cells the b-subunit has evolved to accommodate the interaction with the g-subunit, an associated ATP synthase component only present in the mitochondrial enzyme.