[25] Biogenesis of cytochrome c in Neurospora crassa

Hennig, Bernd; Neupert, Walter

doi:10.1016/0076-6879(83)97138-0

Cited by 64 publications

(37 citation statements)

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“…After synthesis, apocytochrome c has to be imported into the mitochondrion. The import pathway probably does not involve translocation brane (Manella et al, 1987 et al, 1979;Hennig et al, 1984;Nicholson et al, 1987) and biochemical and biophysical studies on apocytochrome c-lipid interactions in model systems (Rietveld et al, 1983(Rietveld et al, , 1986a, complementary models for apocytochrome c import have been proposed. In these models the precursor protein first binds with high affinity, preferentially to the negatively charged lipid component of the outer mitochondrial membrane, followed by penetration and translocation of at least a part of the protein to the opposite interface of the membrane.…”

Section: Lmentioning

confidence: 99%

Specificity of the interaction of amino- and carboxy-terminal fragments of the mitochondrial precursor protein apocytochrome c with negatively charged phospholipids. A spin-label electron spin resonance study

Jordi

Kruijff²,

Marsh

1989

Biochemistry

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ABSTRACT:The contribution of the various regions of the mitochondrial precursor protein apocytochrome c to the interaction of the protein with phosphatidylserine dispersions has been studied with chemically and enzymatically prepared fragments of horse heart apocytochrome c and phospholipids spin-labeled at different positions of the 312-2 chain. Three amino-terminal heme-less peptides, two heme-containing amino-terminal fragments, one central fragment, and three carboxy-terminal fragments were studied. The electron spin resonance spectra of phospholipids spin-labeled at the C5 position of the fatty acid chain indicate that both amino-terminal and carboxy-terminal fragments of the apocytochrome c molecule cause a restriction of motion of the lipids, whereas the heme-containing peptides and protein have less effect. In addition, a second motionally more restricted lipid component, which is observed for apocytochrome c interacting with Interestingly, even a small water-soluble peptide consisting of the 24 carboxy-terminal residues gave rise to a two-component spectrum, with an outer hyperfine splitting of the restricted lipid component of 59 G, indicating a considerable restriction of the chain motion. This suggests that both the carboxy-and amino-terminal parts of the protein penetrate into the center of the bilayer and cause a strong perturbation of the fatty acyl chain motion. The implications of these findings for the mechanism of apocytochrome c translocation across membranes are discussed.

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Section: Lmentioning

confidence: 99%

Specificity of the interaction of amino- and carboxy-terminal fragments of the mitochondrial precursor protein apocytochrome c with negatively charged phospholipids. A spin-label electron spin resonance study

Jordi

Kruijff²,

Marsh

1989

Biochemistry

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“…The number of high-affinity binding sites for apocytochrome c was determined by Scatchard analysis essentially as previously described [22]. Mitochondria, or pellets from digitonin fractions, were suspended at a protein concentration of 1 mg/ml (starting material) in buffer A containing 50 pM deuterohemin, and were incubated for 5 min at 25°C.…”

Section: Subfructionution C?f' Mitochondria With Digitoninmentioning

confidence: 99%

“…The samples were spun at 48 400 x g for 10 min (Beckman JA-20 rotor) and the resulting pellets were resuspended at 0.3 mg protein/ml in 0.25 M sucrose, 50 mM NaCl, 2 mM EDTA, 14 mM 2-mercaptoethanol, 1 % (w/v) bovine serum albumin, 0.02% (w/v) holocytochrome c, 10 mM Mops/KOH, pH 7.2, in silicone-coated glass minivials. The samples were incubated with increasing amounts of ''C-labelled apocytochrome c, prepared by reductive methylation [22], for 20 min at 25 "C. Pellets were re-isolated by centrifugation as above, and the unbound apocytochrome c was measured in the supernatant by radioactivity determination. The pellets were lysed in 0.5 ml of 1% (w/v) SDS, 100 mM TrisiHCl pH 8.0 and the bound apocytochrome c was also quantified by radioactivity determination.…”

Section: Subfructionution C?f' Mitochondria With Digitoninmentioning

confidence: 99%

“…The fractionation of porin, cytochrome c, 20-kDa protein, ADP/ATP carrier and isocitrate dehydrogenase (N and fl subunits) were determined by resolving the samples on SDS/polyacrylamide gels [33] and Western blotting to nitrocellulose membranes [34, 351. The membranes were incubated with a mixture of specific antibodies (against the above-mentioned proteins) and decorated with I4C-labelled protein A (prepared by reductive methylation in the presence of ['4C]formaldehyde as described previously for apocytochrome c [22]). The proteins were visualized by autoradiography and were quantified by cutting out the bands and determining radioactivity.…”

Section: Subfructionution C?f' Mitochondria With Digitoninmentioning

confidence: 99%

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Import of cytochrome c into mitochondria. Cytochrome c heme lyase

1987

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The import of cytochrome c into mitochondria can be resolved into a number of discrete steps. Here we report on the covalent attachment of heme to apocytochrome c by the enzyme cytochrome c heme lyase in mitochondria from Neurospora crassa. A new method was developed to measure directly the linkage of heme to apocytochrome c. This method is independent of conformational changes in the protein accompanying heme attachment. Tryptic peptides of [35S]cysteine‐labelled apocytochrome c, and of enzymatically formed holocytochrome c, were resolved by reverse‐phase HPLC. The cysteine‐containing peptide to which heme was attached eluted later than the corresponding peptide from apocytochrome c and could be quantified by counting 35S radioactivity as a measure of holocytochrome c formation. Using this procedure, the covalent attachment of heme to apocytochrome c, which is dependent on the enzyme cytochrome c heme lyase, could be measured. Activity required heme (as hemin) and could be reversibly inhibited by the analogue deuterohemin. Holocytochrome c formation was stimulated 5–10‐fold by NADH > NADPH > glutathione and was independent of a potential across the inner mitochondrial membrane. NADH was not required for the binding of apocytochrome c to mitochondria and was not involved in the reduction of the cysteine thiols prior to heme attachment. Holocytochrome c formation was also dependent on a cytosolic factor that was necessary for the heme attaching step of cytochrome c import. The factor was a heat‐stable, protease‐insensitive, low‐molecular‐mass component of unknown function. Cytochrome c heme lyase appeared to be a soluble protein located in the mitochondrial intermembrane space and was distinct from the previously identified apocytochrome c binding protein having a similar location. A model is presented in which the covalent attachment of heme by cytochrome c heme lyase also plays an essential role in the import pathway of cytochrome c.

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“…Thus, in yeast cells, cytochrome c can be regulated transcriptionally. Transcriptional regulation of cytochrome c has also been reported for Neurospora crassa, Manduca sexta, and Drosophila melanogaster (22,41). It has been reported that cytochrome c levels can also be subject to posttranscriptional regulation.…”

mentioning

confidence: 89%

Posttranscriptional regulation of cytochrome c expression during the developmental cycle of Trypanosoma brucei.

Torri¹,

Hajduk²

1988

Mol. Cell. Biol.

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We examined the expression of a nucleus-encoded mitochondrial protein, cytochrome c, during the life cycle of Trypanosoma brucei. The bloodstream forms of T. brucei, the long slender and short stumpy trypanosomes, have inactive mitochondria with no detectable cytochrome-mediated respiration. The insect form of T. brucei, the procyclic trypanosomes, has fully functional mitochondria. Cytochrome c is spectrally undetectable in the bloodstream forms of trypanosomes, but during differentiation to the procyclic form, spectrally detected holo-cytochrome c accumulates rapidly. We have purified T. brucei cytochrome c and raised antibodies that react to both holo- and apo-cytochrome c. In addition, we isolated a partial cDNA to trypanosome cytochrome c. An examination of protein expression and steady-state mRNA levels in T. brucei indicated that bloodstream trypanosomes did not express cytochrome c but maintained significant steady-state levels of cytochrome c mRNA. The results suggest that in T. brucei, cytochrome c is developmentally regulated by a posttranscriptional mechanism which prevents either translation or accumulation of cytochrome c in the bloodstream trypanosomes.

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[25] Biogenesis of cytochrome c in Neurospora crassa

Cited by 64 publications

References 14 publications

Specificity of the interaction of amino- and carboxy-terminal fragments of the mitochondrial precursor protein apocytochrome c with negatively charged phospholipids. A spin-label electron spin resonance study

Specificity of the interaction of amino- and carboxy-terminal fragments of the mitochondrial precursor protein apocytochrome c with negatively charged phospholipids. A spin-label electron spin resonance study

Import of cytochrome c into mitochondria. Cytochrome c heme lyase

Posttranscriptional regulation of cytochrome c expression during the developmental cycle of Trypanosoma brucei.

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