plunge-freezing-tool-for-ultrastructural-immunolocalization-studies

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

Université de Bordeaux

The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family, which allows cross-talk between mitochondria, where cell energy is mainly produced, and cytosol, where cell energy is mainly consumed. The members of this family share numerous structural and functional characteristics. Resolution of the atomic structure of the bovine Ancp, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in the movements of the protein to perform translocation of nucleotides from one side of the membrane to the other. They correspond to three prolines located in the odd-numbered transmembrane helices (TMH), Pro-27, Pro-132, and Pro-229. The corresponding residues of the yeast Ancp (Pro-43, Ser-147, and Pro-247) were mutated into alanine or leucine, one at a time and analysis of the various mutants evidenced a crucial role of Pro-43 and Pro-247 during nucleotide transport. Beside, replacement of Ser-147 with proline does not inactivate Ancp and this is discussed in view of the conservation of the three prolines at equivalent positions in the Ancp sequences. These prolines belong to the signature sequences of the mitochondrial carriers and we propose they play a dual role in the mitochondrial ADP/ATP carrier function and biogenesis. Unexpectedly their mutations cause more general effects on mitochondrial biogenesis and morphology, as evidenced by measurements of respiratory rates, cytochrome contents, and also clearly highlighted by fluorescence microscopy.

The transmembrane prolines of the mitochondrial ADP/ATP carrier are involved in nucleotide binding and transport and its biogenesis.

Cell fate and proliferation are tightly linked to the regulation of the mitochondrial energy metabolism. Hence, mitochondrial biogenesis regulation, a complex process that requires a tight coordination in the expression of the nuclear and mitochondrial genomes, has a major impact on cell fate and is of high importance. Here, we studied the molecular mechanisms involved in the regulation of mitochondrial biogenesis through a nutrient-sensing pathway, the Ras-cAMP pathway. Activation of this pathway induces a decrease in the cellular phosphate potential that alleviates the redox pressure on the mitochondrial respiratory chain. One of the cellular consequences of this modulation of cellular phosphate potential is an increase in the cellular glutathione redox state. The redox state of the glutathione disulfide-glutathione couple is a well known important indicator of the cellular redox environment, which is itself tightly linked to mitochondrial activity, mitochondria being the main cellular producer of reactive oxygen species. The master regulator of mitochondrial biogenesis in yeast (i.e. the transcriptional co-activator Hap4p) is positively regulated by the cellular glutathione redox state. Using a strain that is unable to modulate its glutathione redox state (Δglr1), we pinpoint a positive feedback loop between this redox state and the control of mitochondrial biogenesis. This is the first time that control of mitochondrial biogenesis through glutathione redox state has been shown.

cAMP-induced mitochondrial compartment biogenesis: role of glutathione redox state.

The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family (MCF); its members allow metabolic fluxes between mitochondria and the cytosol. The members of the MCF share numerous structural and functional characteristics. Ancp is very specifically inhibited by two classes of compounds, which stabilize the carrier in two different conformations involved in nucleotide transport. Resolution of the atomic structure of the bovine Ancp, in complex with one of its specific inhibitors, is that of the carrier open toward the intermembrane space. To gain insights into the interconversion from one conformation to the other, we introduced point mutations in the yeast carrier at positions Cys73 in the first matrix loop and Tyr97 and Gly298 in transmembrane helices 2 and 6. We demonstrate in this paper that they impair stabilization of the carrier in one conformation or the other, resulting in an almost complete inactivation of nucleotide transport in both cases. The results are discussed on the basis of the atomic structure of the conformation open to the cytosol. These mutant proteins could afford convenient tools for undertaking structural studies of both conformations of the yeast carrier.

Mitochondrial ADP/ATP carrier: preventing conformational changes by point mutations inactivates nucleotide transport activity.

Our understanding of the mechanisms involved in mitochondrial biogenesis has continuously expanded during the last decades, yet little is known about how they are modulated to optimize the functioning of mitochondria. Here, we show that mutations in the ATP binding domain of Bcs1, a chaperone involved in the assembly of complex III, can be rescued by mutations that decrease the ATP hydrolytic activity of the ATP synthase. Our results reveal a Bcs1-mediated control loop in which the biogenesis of complex III is modulated by the energy-transducing activity of mitochondria. Although ATP is well known as a regulator of a number of cellular activities, we show here that ATP can be also used to modulate the biogenesis of an enzyme by controlling a specific chaperone involved in its assembly. Our study further highlights the intramitochondrial adenine nucleotide pool as a potential target for the treatment of Bcs1-based disorders.

The energetic state of mitochondria modulates complex III biogenesis through the ATP-dependent activity of Bcs1.

During the Leishmania life cycle, the flagellum undergoes successive assembly and disassembly of hundreds of proteins. Understanding these processes necessitates the study of individual components. Here, we investigated LdFlabarin, an uncharacterized L. donovani flagellar protein. The gene is conserved within the Leishmania genus and orthologous genes only exist in the Trypanosoma genus. LdFlabarin associates with the flagellar plasma membrane, extending from the base to the tip of the flagellum as a helicoidal structure. Site-directed mutagenesis, deletions and chimera constructs showed that LdFlabarin flagellar addressing necessitates three determinants: an N-terminal potential acylation site and a central BAR domain for membrane targeting and the C-terminal domain for flagellar specificity. In vitro, the protein spontaneously associates with liposomes, triggering tubule formation, which suggests a structural/morphogenetic function. LdFlabarin is the first characterized Leishmania BAR domain protein, and the first flagellum-specific BAR domain protein.

LdFlabarin, a new BAR domain membrane protein of Leishmania flagellum.

In eukaryotic cells, oxidative phosphorylation involves multisubunit complexes of mixed genetic origin. Assembling these complexes requires an organelle-independent synchronizing system for the proper expression of nuclear and mitochondrial genes. Here we show that proper expression of the F1FO ATP synthase (complex V) depends on a cytosolic complex (AME) made of two aminoacyl-tRNA synthetases (cERS and cMRS) attached to an anchor protein, Arc1p. When yeast cells adapt to respiration the Snf1/4 glucose-sensing pathway inhibits ARC1 expression triggering simultaneous release of cERS and cMRS. Free cMRS and cERS relocate to the nucleus and mitochondria, respectively, to synchronize nuclear transcription and mitochondrial translation of ATP synthase genes. Strains releasing asynchronously the two aminoacyl-tRNA synthetases display aberrant expression of nuclear and mitochondrial genes encoding subunits of complex V resulting in severe defects of the oxidative phosphorylation mechanism. This work shows that the AME complex coordinates expression of enzymes that require intergenomic control.

Expression of nuclear and mitochondrial genes encoding ATP synthase is synchronized by disassembly of a multisynthetase complex.

TbFlabarin is the Trypanosoma brucei orthologue of the Leishmania flagellar protein LdFlabarin but its sequence is 33% shorter than LdFlabarin, as it lacks a C-terminal domain that is indispensable for LdFlabarin to localize to the Leishmania flagellum. TbFlabarin is mainly expressed in the procyclic forms of the parasite and localized to the flagellum, but only when two palmitoylable cysteines at positions 3 and 4 are present. TbFlabarin is more strongly attached to the membrane fraction than its Leishmania counterpart, as it resists complete solubilization with as much as 0.5% NP-40. Expression ablation by RNA interference did not change parasite growth in culture, its morphology or apparent motility. Heterologous expression showed that neither TbFlabarin in L. amazonensis nor LdFlabarin in T. brucei localized to the flagellum, revealing non-cross-reacting targeting signals between the two species.

Corinne Blancard

Institut de Biochimie et Génétique Cellulaires,
Centre National de la Recherche Scientifique,
UMR 5095,
Institut de Biochimie et Génétique Cellulaires, Centre National de la Recherche Scientifique, UMR 5095

Université de Bordeaux

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Corinne Blancard

Institut de Biochimie et Génétique Cellulaires,Centre National de la Recherche Scientifique,UMR 5095,Institut de Biochimie et Génétique Cellulaires, Centre National de la Recherche Scientifique, UMR 5095

Université de Bordeaux

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Institut de Biochimie et Génétique Cellulaires,
Centre National de la Recherche Scientifique,
UMR 5095,
Institut de Biochimie et Génétique Cellulaires, Centre National de la Recherche Scientifique, UMR 5095