Preparation of highly phosphorylating mitochondria from the yeastSchizosaccharomyces pombe

Jault, Jean‐Michel; Comte, Jane; Gautheron, D; Pietro, Attilio Di

doi:10.1007/bf00762785

Cited by 12 publications

(15 citation statements)

References 44 publications

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“…This protein can be efficiently imported in vitro into mitochondria of various organisms and represents one of the best characterized precursor proteins. When mitochondria were purified from S. pombe cells according to published procedures (23,37,39), they turned out to be not competent to import pre-Su9DHFR (data not shown). We then isolated mitochondria from S. pombe cells by using a protocol which was adapted from the procedure by which import-competent S. cerevisiae mitochondria are isolated (see Materials and Methods for details).…”

Section: Resultsmentioning

confidence: 99%

Sequential Processing of a Mitochondrial Tandem Protein: Insights into Protein Import in Schizosaccharomyces pombe

et al. 2006

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The sequencing of the genome of Schizosaccharomyces pombe revealed the presence of a number of genes encoding tandem proteins, some of which are mitochondrial components. One of these proteins (pre-Rsm22-Cox11) consists of a fusion of Rsm22, a component of the mitochondrial ribosome, and Cox11, a factor required for copper insertion into cytochrome oxidase. Since in Saccharomyces cerevisiae, Cox11 is physically attached to the mitochondrial ribosome, it was suggested that the tandem organization of Rsm22-Cox11 is used to covalently tie the mitochondrial ribosome to Cox11 in S. pombe. We report here that pre-Rsm22-Cox11 is matured in two subsequent processing events. First, the mitochondrial presequence is removed. At a later stage of the import process, the Rsm22 and Cox11 domains are separated by cleavage of the mitochondrial processing peptidase at an internal processing site. In vivo data obtained using a tagged version of pre-Rsm22-Cox11 confirmed the proteolytic separation of Cox11 from the Rsm22 domain. Hence, the tandem organization of pre-Rsm22-Cox11 does not give rise to a persistent fusion protein but rather might be used to increase the import efficiency of Cox11 and/or to coordinate expression levels of Rsm22 and Cox11 in S. pombe.

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Section: Resultsmentioning

confidence: 99%

Sequential Processing of a Mitochondrial Tandem Protein: Insights into Protein Import in Schizosaccharomyces pombe

et al. 2006

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“…Mitochondria were isolated from C. albicans by spheroplast formation and a mild homogenization procedure. To verify the integrity of the mitochondrial inner membrane, the ''respiratory control rate'' and the uncoupling activity of carbonyl cyanide m-chlorophenylhydrazone (CCCP) were determined for each mitochondrial preparation; these values ranged between 2.5 and 2.7 (9), which are normal values for yeast mitochondria (19). As described previously (9), histatin 5 at a concentration of 33 M inhibits state 2 respiration.…”

Section: Resultsmentioning

confidence: 99%

“…Mitochondria were isolated from C. albicans spheroplasts, essentially as described previously (19). Cells were grown to late logarithmic phase in 1 liter of Sabouraud dextrose broth (Difco), washed once in deionized water, and suspended in 1 ml of Zymolyase buffer containing 50 mM Tris, 10 mM MgCl 2 , and 1.4 M sorbitol (pH 7.5) per g of yeast pellet.…”

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confidence: 99%

The human salivary peptide histatin 5 exerts its antifungal activity through the formation of reactive oxygen species

Helmerhorst

Troxler

Oppenheim

2001

Proc. Natl. Acad. Sci. U.S.A.

213

157

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Previous studies have shown that the human salivary antifungal peptide histatin 5 is taken up by Candida albicans cells and associates intracellularly with mitochondria. The purpose of the present study was to investigate the biological consequence of this specific subcellular targeting. Histatin 5 inhibited respiration of isolated C. albicans mitochondria as well as the respiration of intact blastoconidia in a dose and time-dependent manner. A nearly perfect correlation was observed between histatin-induced inhibition of respiration and cell killing with either logarithmic-or stationary-phase cells, but stationary-phase cells were less sensitive. Because nonrespiring yeast cells are insensitive to histatin 5, the potential mechanistic relationship between histatin 5 interference with the respiratory apparatus and cell killing was explored by using an oxygen radical sensitive probe (dihydroethidium). Fluorimetric measurements showed that histatin 5 induced the formation of reactive oxygen species (ROS) in C. albicans cells as well as in isolated mitochondria and that ROS levels were highly correlated with cell death. In the presence of an oxygen scavenger (L-cysteine), cell killing and ROS formation were prevented. In addition, the membrane-permeant superoxide dismutase mimetic 2,2,6,6-tetramethylpiperidine-N-oxyl, abolished histatin-induced ROS formation in isolated mitochondria. In contrast to histatin 5, the conventional inhibitors of the respiratory chain, sodium cyanide or sodium azide, neither induced ROS nor killed yeast cells. These data provide strong evidence for a comprehensive mechanistic model of histatin-5-provoked yeast cell death in which oxygen radical formation is the ultimate and essential step.

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“…Isolation of Yeast Mitochondria-Mitochondria were isolated from C. albicans spheroplasts essentially as described previously (27). C. albicans (ATCC 10231) were grown to late log phase in 1 liter of Sabouraud dextrose broth (Difco), washed once in 1.2 M sorbitol (Sigma), and suspended in 1.2 M sorbitol supplemented with 50 mM potassium phosphate, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Histatin 5 with Respect to Amphipathicity, Hydrophobicity, and Effects on Cell and Mitochondrial Membrane Integrity Excludes a Candidacidal Mechanism of Pore Formation

Helmerhorst

Hof

Breeuwer³

et al. 2001

Journal of Biological Chemistry

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Histatin 5 is a 24-residue peptide from human saliva with antifungal properties. We recently demonstrated that histatin 5 translocates across the yeast membrane and targets to the mitochondria, suggesting an unusual antifungal mechanism (Helmerhorst, E. J., Breeuwer, P., van't Hof, W., Walgreen-Weterings, E., Oomen, L. C. J. M., Veerman, E. C. I., Nieuw Amerongen, A. V., and Abee, T. The results obtained show that the amphipathic analogs exhibited a high fungicidal activity, a high propensity to form an ␣-helix, dissipated the cytoplasmic transmembrane potential, and uncoupled the respiration of isolated mitochondria, similar to the pore-forming peptide PGLa (Peptide with N-terminal Glycine and C-terminal Leucine-amide). In contrast, histatin 5 and dh-5 showed fewer or none of these features. The difference in these functional characteristics between histatin 5 and dh-5 on the one hand and dhvar1, dhvar4, and PGLa on the other hand correlated well with their predicted affinity for membranes based on hydrophobicity/amphipathicity analysis. These data indicate that the salivary protein histatin 5 exerts its antifungal function through a mechanism other than pore formation.

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Preparation of highly phosphorylating mitochondria from the yeastSchizosaccharomyces pombe

Cited by 12 publications

References 44 publications

Sequential Processing of a Mitochondrial Tandem Protein: Insights into Protein Import in Schizosaccharomyces pombe

Sequential Processing of a Mitochondrial Tandem Protein: Insights into Protein Import in Schizosaccharomyces pombe

The human salivary peptide histatin 5 exerts its antifungal activity through the formation of reactive oxygen species

Characterization of Histatin 5 with Respect to Amphipathicity, Hydrophobicity, and Effects on Cell and Mitochondrial Membrane Integrity Excludes a Candidacidal Mechanism of Pore Formation

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