Jacques Schaeffer

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.

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 non-fermentable conditions. The observation of mitochondria degradation showed that both a selective process and a non-selective process of mitochondria autophagy occurred successively. In a yeast strain inactivated for the gene UTH1, the selective process was not observed.

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.

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 theATP20 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 mitochondrialb-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 subunitg and the first membrane-spanning segment of subunit4 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.

Comparison of the effects of bax‐expression in yeast under fermentative and respiratory conditions: investigation of the role of adenine nucleotides carrier and cytochrome c

A new system for bax‐expression in yeast has been devised to investigate baxs effect under fermentative and respiro‐fermentative conditions. This has allowed us to show unambiguously that the ability of bax to kill yeast is higher under respiratory conditions than under purely fermentative conditions. The extent of killing under respiro‐fermentative conditions (non‐repressive sugars) is intermediate. It has been proposed that the two proteins adenine nucleotides carrier (ANC) and cytochrome c play a crucial role in bax‐induced cell death. We have investigated the effects of deletion of the genes encoding the two proteins on the toxicity induced by bax, using this new system. The absence of ANC did not modify bax‐induced lethality in any way. Moreover, the absence of cytochrome c also did not prevent bax‐induced death. Only the kinetics of lethality were altered. All these effects are prevented by co‐expression of bcl‐x L.

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.

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. (Mol Cell Biochem 267: 157–166, 2004)

The yeast Rvs161 and Rvs167 proteins are involved in secretory vesicles targeting the plasma membrane and in cell integrity

The Rvs161 and Rvs167 proteins are known to play a role in actin cytokeleton organization and endocytosis. Moreover, Rvs167p functionally interacts with the myosin Myo2p. Therefore, we explored the involvement of the Rvs proteins in vesicle traffic and in cell integrity. The rvs mutants accumulate late secretory vesicles at sites of membrane and cell wall construction. They are synthetic‐lethal with the slt2/mpk1 mutation, which affects the MAP kinase cascade controlled by Pkc1p and is required for cell integrity. The phenotype of the double mutants is close to that described for the pkc1 mutant. Synthetic defects for growth are also observed with mutation in KRE6, a gene coding for a glucan synthase, required for cell wall construction. These data support the idea that the Rvs proteins are involved in the late targeting of vesicles whose cargoes are required for cell wall construction. Copyright

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.

A second DNA polymerase activity in yeast mitochondria

In eukaryotic cells, there is much evidence to indicate that the replication of the mitochondrial genome is carried out by a specific DNA polymerase named DNA polymerase gamma. In the yeast S. cerevisiae, a DNA polymerase gamma has been partially purified and the gene encoding the catalytic subunit identified. The characteristics of this enzyme are the same as those found in higher eukaryotes, except for the requirement for a higher magnesium concentration. During a purification procedure of yeast mitochondrial DNA polymerase, we have isolated a second DNA polymerase activity. Using different approaches ive have ruled out the possibility of nuclear contamination or a product of proteolysis. From its properties, this new DNA polymerase activity seems to be different from any yeast DNA polymerase. This new mitochondrial DNA polymerase activity provides evidence that the animal model of mitochondrial DNA replication cannot be generalized. The presence of two DNA polymerases in yeast mitochondria could reflect a different replication or repair mechanism.