scholarly journals Cmc1p Is a Conserved Mitochondrial Twin CX9C Protein Involved in Cytochrome c Oxidase Biogenesis

2008 ◽  
Vol 28 (13) ◽  
pp. 4354-4364 ◽  
Author(s):  
Darryl Horn ◽  
Hassan Al-Ali ◽  
Antoni Barrientos
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Superoxide Dismutase ◽  
Cytochrome C Oxidase ◽  
Mitochondrial Enzymes ◽  
Intermembrane Space ◽  
Mitochondrial Matrix ◽  
Copper Chaperones ◽  
Copper Trafficking ◽  
Mitochondrial Inner Membrane ◽  
Copper Incorporation

ABSTRACT Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by metallochaperone proteins which probably use copper from a mitochondrial matrix-localized pool. Here we describe a novel conserved mitochondrial metallochaperone-like protein, Cmc1p, whose function affects both COX and Sod1p. In Saccharomyces cerevisiae, Cmc1p localizes to the mitochondrial inner membrane facing the intermembrane space. Cmc1p is essential for full expression of COX and respiration, contains a twin CX9C domain conserved in other COX assembly copper chaperones, and has the ability to bind copper(I). Additionally, mutant cmc1 cells display increased mitochondrial Sod1p activity, while CMC1 overexpression results in decreased Sod1p activity. Our results suggest that Cmc1p could play a direct or indirect role in copper trafficking and distribution to COX and Sod1p.

scholarly journals Mitochondrial Superoxide Dismutase and Yap1p Act as a Signaling Module Contributing to Ethanol Tolerance of the Yeast Saccharomyces cerevisiae

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Anna N. Zyrina ◽  
Ekaterina A. Smirnova ◽  
Olga V. Markova ◽  
Fedor F. Severin ◽  
Dmitry A. Knorre
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hydrogen Peroxide ◽  
Superoxide Dismutase ◽  
Ethanol Tolerance ◽  
Yeast Cells ◽  
Ethanol Stress ◽  
Mitochondrial Enzymes ◽  
Mitochondrial Superoxide ◽  
Mitochondrial Superoxide Dismutase

ABSTRACT There are two superoxide dismutases in the yeast Saccharomyces cerevisiae—cytoplasmic and mitochondrial enzymes. Inactivation of the cytoplasmic enzyme, Sod1p, renders the cells sensitive to a variety of stresses, while inactivation of the mitochondrial isoform, Sod2p, typically has a weaker effect. One exception is ethanol-induced stress. Here we studied the role of Sod2p in ethanol tolerance of yeast. First, we found that repression of SOD2 prevents ethanol-induced relocalization of yeast hydrogen peroxide-sensing transcription factor Yap1p, one of the key stress resistance proteins. In agreement with this, the levels of Trx2p and Gsh1p, proteins encoded by Yap1 target genes, were decreased in the absence of Sod2p. Analysis of the ethanol sensitivities of the cells lacking Sod2p, Yap1p, or both indicated that the two proteins act in the same pathway. Moreover, preconditioning with hydrogen peroxide restored the ethanol resistance of yeast cells with repressed SOD2. Interestingly, we found that mitochondrion-to-nucleus signaling by Rtg proteins antagonizes Yap1p activation. Together, our data suggest that hydrogen peroxide produced by Sod2p activates Yap1p and thus plays a signaling role in ethanol tolerance. IMPORTANCE Baker's yeast harbors multiple systems that ensure tolerance to high concentrations of ethanol. Still, the role of mitochondria under severe ethanol stress in yeast is not completely clear. Our study revealed a signaling function of mitochondria which contributes significantly to the ethanol tolerance of yeast cells. We found that mitochondrial superoxide dismutase Sod2p and cytoplasmic hydrogen peroxide sensor Yap1p act together as a module of the mitochondrion-to-nucleus signaling pathway. We also report cross talk between this pathway and the conventional retrograde signaling cascade activated by dysfunctional mitochondria.

scholarly journals Absence of the iron-containing superoxide dismutase in mitochondria from mustard (Brassica campestris)

1981 ◽  
Vol 195 (1) ◽  
pp. 229-233 ◽  
Author(s):  
M L Salin ◽  
S M Bridges
Keyword(s):  
Superoxide Dismutase ◽  
Brassica Campestris ◽  
Polyacrylamide Gel ◽  
Superoxide Dismutases ◽  
Intermembrane Space ◽  
Mitochondrial Matrix ◽  
Mature Leaves ◽  
Etiolated Seedlings

Mitochondria were isolated from mature leaves as well as etiolated seedlings of Brassica campestris (mustard), a eukaryote previously shown to possess the iron-containing isoenzyme of superoxide dismutase. On the basis of KCN- and H2O2-sensitivity, and on polyacrylamide-gel analysis, only the cuprozinc and mangano superoxide dismutases were found in mitochondria. The iron-containing enzyme was absent. The mangano enzyme was found in the mitochondrial matrix, whereas the cuprozinc enzyme appeared to be localized in the intermembrane space.

scholarly journals The Yeast Aac2 Protein Exists in Physical Association with the Cytochromebc1-COX Supercomplex and the TIM23 Machinery

2008 ◽  
Vol 19 (9) ◽  
pp. 3934-3943 ◽  
Author(s):  
Mary K. Dienhart ◽  
Rosemary A. Stuart
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Affinity Purification ◽  
Yeast Saccharomyces Cerevisiae ◽  
Present Evidence ◽  
Cytochrome Bc ◽  
Saccharomyces Cerevisiae Mitochondria ◽  
Mitochondrial Inner Membrane ◽  
Physical Association

The ADP/ATP carrier (AAC) proteins play a central role in cellular metabolism as they facilitate the exchange of ADP and ATP across the mitochondrial inner membrane. We present evidence here that in yeast (Saccharomyces cerevisiae) mitochondria the abundant Aac2 isoform exists in physical association with the cytochrome c reductase (cytochrome bc1)-cytochrome c oxidase (COX) supercomplex and its associated TIM23 machinery. Using a His-tagged Aac2 derivative and affinity purification studies, we also demonstrate here that the Aac2 isoform can be affinity-purified with other AAC proteins. Copurification of the Aac2 protein with the TIM23 machinery can occur independently of its association with the fully assembled cytochrome bc1-COX supercomplex. In the absence of the Aac2 protein, the assembly of the cytochrome bc1-COX supercomplex is perturbed, whereby a decrease in the III2-IV2assembly state relative to the III2-IV form is observed. We propose that the association of the Aac2 protein with the cytochrome bc1-COX supercomplex is important for the function of the OXPHOS complexes and for the assembly of the COX complex. The physiological implications of the association of AAC with the cytochrome bc1-COX-TIM23 supercomplex are also discussed.

scholarly journals Subcellular distribution of enzymes determined by rapid digitonin fractionation of isolated hepatocytes

1980 ◽  
Vol 186 (2) ◽  
pp. 423-429 ◽  
Author(s):  
Alvin M. Janski ◽  
Neal W. Cornell
Keyword(s):  
Minimum Time ◽  
Mitochondrial Enzymes ◽  
Intermembrane Space ◽  
Mitochondrial Matrix ◽  
Atp Citrate Lyase ◽  
Rapid Separation ◽  
Citrate Lyase ◽  
Ionic Detergent ◽  
Cytosolic Enzymes ◽  
Enzyme Markers

Conditions were determined for rapid separation of cytosolic and mitochondrial compartments by digitonin fractionation of rat hepatocytes. The minimum time required for separation of mitochondrial and cytosolic enzyme markers decreased rapidly with increasing temperature. Kyro EOB, a non-ionic detergent, increases the release of cytosolic enzymes, particularly at lower temperatures. Experimental procedures are described for greater than 90% release of cytosolic enzymes and less than 2% release of mitochondrial enzymes in 3s. By using appropriate concentrations of digitonin and Kyro EOB in a fractionation medium maintained at 1°C and a minimum time of exposure to the medium, nearly separate patterns of release were obtained for enzyme markers for the cytosol, mitochondrial matrix and mitochondrial intermembrane space. The distribution of enzymes that exist in more than one of these compartments was quantified by comparing their rates of release with those of marker enzymes. The cytosol/mitochondrial-matrix distributions for such enzymes in hepatocytes from starved rats were 16%/84% for aspartate aminotransferase, 34%/66% for fumarase and 77%/23% for ATP citrate lyase. In hepatocytes from rats that were induced to synthesize ATP citrate lyase by starvation and re-feeding, the ratio had increased to 95%/5%. The maximum cytosol/intermembrane-space ratio for adenylate kinase was 8%/92%. A procedure is also described for treating commercial digitonin that increases its solubility in water from about 1mg/ml to more than 800mg/ml.

scholarly journals Pet191 Is a Cytochrome c Oxidase Assembly Factor in Saccharomyces cerevisiae

2008 ◽  
Vol 7 (8) ◽  
pp. 1427-1431 ◽  
Author(s):  
Oleh Khalimonchuk ◽  
Kevin Rigby ◽  
Megan Bestwick ◽  
Fabien Pierrel ◽  
Paul A. Cobine ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Disulfide Linkages ◽  
Assembly Factor ◽  
Oligomeric Complex

ABSTRACT The twin-Cx9C motif protein Pet191 is essential for cytochrome c oxidase maturation. The motif Cys residues are functionally important and appear to be present in disulfide linkages within a large oligomeric complex associated with the mitochondrial inner membrane. The import of Pet191 differs from that of other twin-Cx9C motif class of proteins in being independent of the Mia40 pathway.

scholarly journals Deletion of the Leader Peptide of the Mitochondrially Encoded Precursor of Saccharomyces cerevisiae Cytochrome c Oxidase Subunit II

Genetics ◽  
1997 ◽  
Vol 145 (4) ◽  
pp. 903-910 ◽  
Author(s):  
A Thomas Torello ◽  
Michael H Overholtzer ◽  
Vicki L Cameron ◽  
Nathalie Bonnefoy ◽  
Thomas D Fox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Cytochrome B ◽  
Mitochondrial Gene ◽  
Leader Peptide ◽  
Intermembrane Space ◽  
Gene Encoding ◽  
Amino Terminal ◽  
Oxidase Subunit

Cytochrome c oxidase subunit II (Cox2p) of Saccharomyces cerevisiae is synthesized within mitochondria as a precursor, pre-Cox2p. The 15-amino acid leader peptide is processed after export to the intermembrane space. Leader peptides are relatively unusual in mitochondrially coded proteins: indeed mammalian Cox2p lacks a leader peptide. We generated two deletions in the S. cerevisiae COX2 gene, removing either the leader peptide (cox2-20) or the leader peptide and processing site (cox2-21) without altering either the promoter or the mRNA-specific translational activation site. When inserted into mtDNA, both deletions substantially reduced the steady-state levels of Cox2p and caused a tight nonrespiratory phenotype. A respiring pseudorevertant of the cox2-20 mutant was heteroplasmic for the original mutant mtDNA and a ρ–-mtDNA whose deletion fused the first 251 codons of the mitochondrial gene encoding cytochrome b to the cox2-20 sequence. The resulting fusion protein was processed to yield functional Cox2p. Thus, the presence of amino-terminal cytochrome b sequence bypassed the need for the pre-Cox2p leader peptide. We propose that the pre-Cox2p leader peptide contains a targeting signal necessary for membrane insertion, without which it remains in the matrix and is rapidly degraded.

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