Purification of the Cytochrome bc1 Complex from Yeast

Pálsdóttir, Hildur; Hunte, Carola

doi:10.1016/b978-012361776-7/50012-7

Cited by 9 publications

(10 citation statements)

References 28 publications

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“…, respectively, which is comparable with published results (33,39,40). Even though the purification of the complexes utilized detergents, tightly bound lipids were previously found associated with highly purified preparations (41).…”

supporting

confidence: 83%

“…Mitochondria (80 mg of protein) were resuspended in 10 ml of lysis buffer containing 2% dodecyl ␤-D-maltoside (DDM, Anatrace), 50 mM potassium acetate, 10% glycerol, 1:50 volume of protease inhibitor mixture for fungal and yeast cells (Sigma), 1.5 mM phenylmethylsulfonyl fluoride, and 30 mM HEPES-KOH, pH 7.4, for 90 min at 4°C. After centrifugation (145,000 ϫ g, 20 min), the supernatant was loaded on a DEAE CL-6B column (GE, 4 ml of resin) for CIII immobilization (33). The flowthrough was collected and saved for CIV purification (see below).…”

Section: Methodsmentioning

confidence: 99%

“…1) as in the mitochondrial inner membrane (29). The higher amount of CL in CIII than was determined in the crystal structure is a result of the less delipidation of this enzyme because only one step of the purification procedure was used instead of the two steps as described previously (33). We chose not to use the second step, which resulted in the loss of subunit Qcr10p.…”

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confidence: 99%

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Cardiolipin-dependent Reconstitution of Respiratory Supercomplexes from Purified Saccharomyces cerevisiae Complexes III and IV

Bazán¹,

Mileykovskaya²,

Mallampalli³

et al. 2013

Journal of Biological Chemistry

127

108

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Background: Cardiolipin is required for in vivo respiratory supercomplex formation in Saccharomyces cerevisiae. Results: Supercomplex III 2 IV 2 reconstitution from purified complexes III and IV was dependent on addition of cardiolipin over their tightly bound amounts. Electron microscopy confirmed supercomplex organization. Conclusion: Supercomplex III 2 IV 2 formation is absolutely contingent on cardiolipin presence in the membrane. Significance: This minimal system provides understanding of lipid-dependent supercomplex dynamics in vivo.

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confidence: 83%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Cardiolipin-dependent Reconstitution of Respiratory Supercomplexes from Purified Saccharomyces cerevisiae Complexes III and IV

Bazán¹,

Mileykovskaya²,

Mallampalli³

et al. 2013

Journal of Biological Chemistry

127

108

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“…(c and d) The activity of the cytochrome bc 1 complex (c) and the cytochrome c oxidase (d) was determined in submitochondrial particles prepared from wild-type, psd1 Δ, and psd1 Δ psd2 Δ yeast cells as described earlier 83,84 . Ubiquinol-dependent cytochrome c reduction was measured as described by Palsdottir and Hunte 84 using 3 μg protein (submitochondrial particles), 50 μM horse heart cytochrome c , and 80 μM decylubiquinol for 1 ml assay volume (40 mM potassium phosphate buffer, pH 7.4, 1 mM NaN 3 , and 0.05 % β- d -undecylmaltoside). Reduction of cytochrome c was monitored at 550 nm and the activity was calculated with an extinction coefficient of 19.4 mM − 1 cm − 1 .…”

Section: Figmentioning

confidence: 99%

Phosphatidylethanolamine and Cardiolipin Differentially Affect the Stability of Mitochondrial Respiratory Chain Supercomplexes

Böttinger

Horvath

Kleinschroth

et al. 2012

Journal of Molecular Biology

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193

148

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The mitochondrial inner membrane contains two non-bilayer‐forming phospholipids, phosphatidylethanolamine (PE) and cardiolipin (CL). Lack of CL leads to destabilization of respiratory chain supercomplexes, a reduced activity of cytochrome c oxidase, and a reduced inner membrane potential Δψ. Although PE is more abundant than CL in the mitochondrial inner membrane, its role in biogenesis and assembly of inner membrane complexes is unknown. We report that similar to the lack of CL, PE depletion resulted in a decrease of Δψ and thus in an impaired import of preproteins into and across the inner membrane. The respiratory capacity and in particular the activity of cytochrome c oxidase were impaired in PE-depleted mitochondria, leading to the decrease of Δψ. In contrast to depletion of CL, depletion of PE did not destabilize respiratory chain supercomplexes but favored the formation of larger supercomplexes (megacomplexes) between the cytochrome bc1 complex and the cytochrome c oxidase. We conclude that both PE and CL are required for a full activity of the mitochondrial respiratory chain and the efficient generation of the inner membrane potential. The mechanisms, however, are different since these non-bilayer‐forming phospholipids exert opposite effects on the stability of respiratory chain supercomplexes.

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“…Transformed cells were cultured at 30 °C in synthetic complete (SC) medium in the presence of 2% (w/v) glucose without uracil [45]. Cultures were diluted to an A 600 nm of 0.4 in SC medium containing 2% (w/v) galactose and growth was continued for 12 h. Cells were harvested by centrifugation and immediately used for the isolation of total membranes [46] or mitochondria [28,47]. Microsomes were separated from mitochondria by centrifugation of the resulting supernatant at 100 000 g for 45 min at 4 °C (Ti45, Beckman Coulter, Fullerton, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Switching of the homooligomeric ATP‐binding cassette transport complex MDL1 from post‐translational mitochondrial import to endoplasmic reticulum insertion

et al. 2007

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ATP-binding cassette (ABC) transporters belong to a large family of membrane proteins found in all three kingdoms of life. The chemical energy of ATP is used to drive uphill transport of a broad range of solutes across membranes [1][2][3]. ABC transporters have a conserved domain organization consisting of two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The TMDs form a translocation pore, whereas the NBDs catalyze ATP hydrolysis.The ABC half-transporter multidrug resistance like protein 1 (MDL1), composed of a TMD followed by a NBD, is located in the inner mitochondrial membrane (IMM) of Saccharomyces cerevisiae. It has been suggested to be involved in the export of 6-mer to 20-mer peptides, derived from proteolysis of nonassembled inner membrane proteins by the m-AAA (i.e. matrixoriented ATPase associated with a variety of cellular activities) protease [4]. It has been further reported that MDL1 mediates resistance against oxidative stress and can partially complement the function of ABC transporter of mitochondria (ATM) 1 [5]. Deletion of ATM1 in S. cerevisiae results in a severe growth defect because ATM1 is essential for the biogenesis of cytosolic iron-sulfur (Fe-S) proteins [6]. The ATP-binding cassette transporter MDL1 of Saccharomyces cerevisiae has been implicated in mitochondrial quality control, exporting degradation products of misassembled respiratory chain complexes. In the present study, we identified an unusually long leader sequence of 59 amino acids, which targets MDL1 to the inner mitochondrial membrane with its nucleotidebinding domain oriented to the matrix. By contrast, MDL1 lacking this leader sequence is directed into the endoplasmic reticulum membrane with the nucleotide-binding domain facing the cytosol. Remarkably, in both targeting routes, the ATP-binding cassette transporter maintains its intrinsic properties of membrane insertion and assembly, leading to homooligomeric complexes with similar activities in ATP hydrolysis. The physiological consequences of both targeting routes were elucidated in cells lacking the mitochondrial ATP-binding cassette transporter ATM1, which is essential for biogenesis of cytosolic iron-sulfur proteins. The mitochondrial MDL1 complex can complement ATM1 function, whereas the endoplasmic reticulumtargeted version, as well as MDL1 mutants deficient in ATP binding and hydrolysis, cannot overcome the Datm1 growth phenotype.Abbreviations ABC, ATP-binding cassette; ATM, ABC transporter of mitochondria; ER, endoplasmic reticulum; 5-FOA, 5-fluoroorotic acid; IMM, inner mitochondrial membrane; MDL1, multidrug resistance like protein 1; MTS, mitochondrial targeting signal; NBD, nucleotide-binding domain; SC, synthetic complete; TIM, translocase of the inner mitochondrial membrane; TOM, translocase of the outer mitochondrial membrane; TMD, transmembrane domain.

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Purification of the Cytochrome bc1 Complex from Yeast

Cited by 9 publications

References 28 publications

Cardiolipin-dependent Reconstitution of Respiratory Supercomplexes from Purified Saccharomyces cerevisiae Complexes III and IV

Cardiolipin-dependent Reconstitution of Respiratory Supercomplexes from Purified Saccharomyces cerevisiae Complexes III and IV

Phosphatidylethanolamine and Cardiolipin Differentially Affect the Stability of Mitochondrial Respiratory Chain Supercomplexes

Switching of the homooligomeric ATP‐binding cassette transport complex MDL1 from post‐translational mitochondrial import to endoplasmic reticulum insertion

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