Lipophilic Proteins Encoded by Mitochondrial and Nuclear Genes in <i>Neurospora crassa</i>

Küntzel, Hans; Pienia̧żzek, Norman J.; Pienia̧źek, Danuta; Leister, Dennis E.

doi:10.1111/j.1432-1033.1975.tb04169.x

Cited by 20 publications

(4 citation statements)

References 25 publications

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“…The proteins extractable from whole mitochondria of Neurosporu crussu with chloroform/methanol (2/1) have been resolved by dodecylsulfate/Sephadex G-200 filtration into a non-aggregating small protein species (apparent molecular weight around 8000) of extramitochondrial origin, and a heavy dodecylsulfateresistant aggregate of a small intramitrochondrially synthesized protein of about the same apparent molecular weight [36]. The first fraction probably corresponds to the extramitochondrially synthesized dicyclohexylcarbodiimide-binding proteolipid (apparent molecular weight around 8000) isolated from whole mitochondrial membranes or from the solubilized ATPase complex by a similar extraction method [32].…”

Section: Discussionmentioning

confidence: 99%

Identification of an Intramitochondrially Synthesized Proteolipid Associated with the Mitochondrial ATPase Complex as the Product of a Mitochondrial Gene Determining Oligomycin Resistance in Aspergillus nidulans

et al. 1977

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An extranuclear oligomycin‐resistant mutant of Aspergillus nidulans was isolated and biochemically analyzed. The mutant grows slowly in the absence of oligomycin and contains an excess of cytochrome c. The ATPase complex solubilized from mutant mitochondria with Triton X‐100 is 80 times more resistant to oligomycin than the enzyme of the parental strain. The enzyme complexes of both strains were further purified to electrophoretic homogeneity and resolved by dodecylsulfate gel electrophoresis in the presence of 8 M urea into 16 polypeptide bands of apparent molecular weights between 65000 and 6000. Four proteins of apparent molecular weights 43000, 21000, 11000 and 6000 are synthesized on cycloheximide‐resistant mitochondrial ribosomes. The smallest subunit is selectively extracted from the purified enzyme by neutral chloroform/methanol (2/1). A proteolipid of identical electrophoretic mobility and site of synthesis is the major component of proteins extractable with the same solvent from whole mitochondrial membranes. This protein has been isolated from the mutant and its parental strain, and purified to apparent homogeneity. The proteins from both strains start with N‐formyl‐methionine and end with valine. However, a second internal methionine residue present in the wild‐type protein is absent, together with some other amino acids, from the mutant protein. It is concluded that this small lipophilic protein is coded by a mitochondrial gene determining oligomycin resistance, is synthesized on mitochondrial ribosomes and is associated with the membrane sector of the ATPase complex. The mutational alteration of the protein not only confers oligomycin resistance to the ATPase complex but also causes a structural alteration of the mitochondrial inner membrane.

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Section: Discussionmentioning

confidence: 99%

Identification of an Intramitochondrially Synthesized Proteolipid Associated with the Mitochondrial ATPase Complex as the Product of a Mitochondrial Gene Determining Oligomycin Resistance in Aspergillus nidulans

et al. 1977

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“…Similar results have been reported with other mitochondrial systems, showing that the major polypeptides encoded by the mitochondrial genome are characterized by apparent M I of 8000 -10 000. On the other hand, there are examples of high molecular weight products of mitochondrial protein synthesis obtained under appropriate conditions which are capable of yielding small peptides by various chemical and physical treatments [24,Tzagoloff and Akai [30] observed in the yeast Saccharomyces cerevisiae a conversion of a major mitochondrial product of M , 45000 into a peptide of M , 7500 by treatment with chloroform/ methanol or sodium dodecylsulfate at alkaline pH. This peptide was later claimed to be subunit 9 of the ATPase complex [8].…”

Section: Discussionmentioning

confidence: 99%

“…Burke and Beattie [33] also mentioned the reduction in size, subsequent to lyophilisation, of high molecular weight products synthetized in rat liver mitochondria. In Neurospora crassu, Kuntzel et al [24] showed that 30% of the translation products of the mitochondrial DNA in vitro are aggregates of small polypeptides of M I around 8000, which can be disaggregated by mild oxidation with performic acid. Brambl and Hanschin [34] showed that treatment of fungal mitochondria with trichloroacetic acid, before extraction with detergent, caused conversion of mitochondrial products of high molecular weight to peptides of M I 8400.…”

Section: Discussionmentioning

confidence: 99%

The Dicyclohexylcarbodiimide-Binding Protein of Rat Liver Mitochondria as a Product of the Mitochondrial Protein Synthesis

1978

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A product of mitochondrial protein synthesis in rat liver mitochondria, characterized by a low molecular weight ( M , < 10000) and an unusually high hydrophobicity, has been identified as the dicyclohexylcarbodiimide-binding protein and as a peptide of the hydrophobic sector of the mitochondrial ATPase complex. The purified protein still possesses the ability to bind dicyclohexylcarbodiimide.Since the first report by Beechey et al. [l] that dicyclohexylcarbodiimide inhibits oxidative phosphorylation, there have been a number of reports on the isolation from mitochondria [2], chloroplasts [3] and bacteria [4,5j, of a small molecular weight protein capable of binding dicyclohexylcarbodiimide irreversibly and probably covalently. The dicyclohexylcarbodiimide-binding protein appears to be a subunit of the hydrophobic sector of the ATPase complex [6]. It is a proteolipid soluble in chloroform/methanol. In spite of a large amount of work on the chemical characterization of the dicyclohexylcarbodiimide-binding protein, little information is available on the biosynthesis of this protein in mitochondria. In Neurospora crassa, the dicyclohexylcarbodiimide-binding protein is synthetized outside of mitochondria on cytoplasmic ribosomes [7j. On the other hand, it has been suggested that in the yeast, Saccharomyces cerevisiae, the smallest component of the oligomycin-sensitive ATPase, subunit 9, which is synthetized on mitochondrial ribosomes, could be the component which interacts with rutamycin and dicyclohexylcarbodiimide [8].In a previous study, two low-molecular-weight products of mitochondrial protein synthesis were isolated from a chloroform/methanol extract of a trichloroacetic acid precipitate of rat liver mitochondria. They were characterized by an unusual hydrophobicity reflected by their behaviour on paper chromatography. Because of their chromatographic properties they were designated as fast-running products. The two products were further separated by thin-layer chromatography and referred to as fast-running Enzyme. ATP phosphohydrolase or ATPase (EC 3.6.1.3) product A and fast-running product B respectively [9]. In this paper, we show that fast-running product B of the mitochondrial protein synthesis is the dicyclohexylcarbodiimide-binding protein. Some of the data have been presented in an abstract form [lo]. MATERIALS AND METHODS Chemicals Procedures for Mitochondria1 PreparationsRat liver mitochondria were isolated from Wistar rats [ l l ] and resuspended in 0.25 M sucrose, 10 mM Tris-HC1, pH 7.4. They were washed twice in 0.25 M sucrose, 1 mM EDTA and 10 mM Tris-HCI, pH 7.4.Beef heart mitochondria were prepared according to Smith [12]. Submitochondrial particles were prepared by sonication of rat liver or beef heart mitochondria after dilution to a concentration of 10-15 mg protein in 0.25 M sucrose, 1 mM EDTA and 10 mM Tris-HC1, pH 7.4. The mitochondrial suspension was exposed to sonic oscillations in a Branson sonifier at 100 W for six periods of 30 s each, separated by 1-min intervals. The temperature ...

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“…Performic acid has been reported to dissaggregate a mitochondrial protein extracted from Neurospora crassa with chloroform/methanol into units of a molecular weight of about 8000 [23]. We incubated the isolated oligomycin-and dicyclohexylcarbodiimide-sensitive ATPase with [ 14C]dicyclohexylcarbodiimide and then treated the preparation with performic acid at 50°C (see Materials and Methods).…”

Section: Re Cons T I T U T Edmentioning

confidence: 99%

A Study of the Dicyclohexylcarbodiimide-Binding Component of the Mitochondrial ATPase Complex from Beef Heart

Glaser

Norling

Ernster

2005

European Journal of Biochemistry

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1. The binding of [14C]dicyclohexylcarbodiimide to membrane proteins of beef heart mitochondria has been investigated using dodecylsulphate/polyacrylamide gel electrophoresis. Upon incubation of submitochondrial particles with low concentrations of dicyclohexylcarbodiimide (5 nmol;mg protein) radioactivity was incorporated into three components with apparent molecular weights of 30000, 18000 and less than 6500. Only the two smaller components were found to be extracted into chloroform/methanol. The same two components were labelled when the isolated ATPase complex or a reconstituted FoFl system was incubated with low concentrations of dicyclohexylcarbodiimide. High concentrations of dicyclohexylcarbodiimide (20 --100 nmol/mg protein) resulted in binding to several mitochondrial proteins.2. The maximal amount of dicyclohexylcarbodiimide which can bind to submitochondrial particles, the isolated ATPase complex, and the reconstituted FoFl system was found to exceed the amount required for maximal inhibition of the ATPase activity by several-fold. The distribution of the bound [14C]dicyclohexylcarbodiimide between the different dicyclohexylcarbodiimide-binding components was investigated as a function of dicyclohexylcarbodiimide concentration. The smallest and largest components revealed a high affinity for dicyclohexylcarbodiimidebinding which paralleled the inhibition of ATPase activity. The intermediate component had a markedly lower affinity for dicyclohexylcarbodiimide-binding.3. The larger dicyclohexylcarbodiimide-binding component of the isolated ATPase complex can be converted into the smaller component by treatment of the ATPase complex with performic acid. Partial conversion can also be achieved by extraction of the band from the dodecylsulphate-polyacrylamide gel after electrophoresis, followed by re-electrophoresis. The observations suggest that the larger component may be an oligomer of the smaller one.4. Using concentrations of oligomycin and dicyclohexylcarbodiimide which were equal to or greater than those required for maximal inhibition of the ATPase activity, oligomycin was found to diminish the binding of [14C]dicyclohexylcarbodiimide to both dicyclohexylcarbodiimide-binding components of the isolated ATPase complexThe oligomycin-and dicyclohexylcarbodiimide-sensitive ATPase system is responsible for mitochondrial ATP synthesis during respiration [I]. It is composed of two structurally and functionally distinct units: FI, a hydrophilic, periferal membrane component ; Fo, a hydrophobic, membrane-integrated part. The soluble portion F1 exhibits ATPase activity, while the hydrophobic portion Fo is involved in the transport ofprotons across themitochondrial inner membrane. Oligomycin and dicyclohexylcarbodiimide have been found lo act on Fo, and to block the enzymic activity of the whole complex as well a s the proton translocation through Fo.Dicyclohexylcarbodiimide reacts irreversibly and quite specifically with a low molecular weight polypeptide as first shown with beef heart mitochondria [2]. This...

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Lipophilic Proteins Encoded by Mitochondrial and Nuclear Genes in Neurospora crassa

Cited by 20 publications

References 25 publications

Identification of an Intramitochondrially Synthesized Proteolipid Associated with the Mitochondrial ATPase Complex as the Product of a Mitochondrial Gene Determining Oligomycin Resistance in Aspergillus nidulans

Identification of an Intramitochondrially Synthesized Proteolipid Associated with the Mitochondrial ATPase Complex as the Product of a Mitochondrial Gene Determining Oligomycin Resistance in Aspergillus nidulans

The Dicyclohexylcarbodiimide-Binding Protein of Rat Liver Mitochondria as a Product of the Mitochondrial Protein Synthesis

A Study of the Dicyclohexylcarbodiimide-Binding Component of the Mitochondrial ATPase Complex from Beef Heart

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