Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae.

Velours, Jean; Esparza, María Martínez; Hoppe, Jürgen; Sebald, Walter; Guerin, Bemard

doi:10.1002/j.1460-2075.1984.tb01785.x

Cited by 70 publications

(29 citation statements)

References 42 publications

(42 reference statements)

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“…In contrast, Y8, both as the authentic in vivo product (lane 1) or the in vitro translation product of the NAP8 gene (lane 3), shows mobility consistent with its actual size of 5.8 kDa [12, 131 also paralleling the size estimated by gel permeation chromatography [25]. One should note in passing that the anomalous mobility of yeast subunit 9 has led to subunit 8 being incorrectly referred to as a 10-kDa protein [25 -281, merely because the more rapidly moving subunit 9 was erroneously taken as a reliable 8-kDa size/mobility standard.…”

Section: Synthesis Of Nap9mentioning

confidence: 85%

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Reprogrammed expression of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae

Farrell¹,

Gearing²,

Nagley³

1988

European Journal of Biochemistry

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A synthetic gene has been designed and constructed by total chemical synthesis as a first step in the functional relocation from the mitochondrion to the nucleus of a gene encoding subunit 9 of the yeast mitochondrial ATPase complex. This gene (NAP9) incorporates codons frequently used in nuclear genes of Saccharomyces cerevisiae and additionally includes a series of unique restriction enzyme cleavage sites to facilitate future systematic manipulations of the gene and its protein product. Following the expression of the NAP9 gene by transcription and translation in vitro, a radiolabelled protein was produced which displayed a gel electrophoretic mobility and solubility in chloroform/methanol characteristic of the authentic subunit 9 proteolipid encoded in vivo by the mitochondrial olil gene. In order to achieve import into mitochondria of yeast subunit 9, a fusion was made between the NAP9 gene and DNA encoding the cleavable presequence of the nuclearly encoded precursor to subunit 9 from Neurospora crassa. Following expression in vitro, the resultant fusion protein was imported and appropriately processed by isolated yeast mitochondria. The import of yeast subunit 9 was less efficient than that observed in parallel import experiments with yeast subunit 8 attached to the same presequence or with the naturally occurring intact N . crassa subunit 9 precursor. Yeast subunit 9 lacking a leader sequence is not imported into mitochondria but, unlike subunit 8, it does not embed itself into the outer membrane, in spite of its highly hydrophobic character.FoF1-ATPase complexes in energy-transducing membranes of mitochondria, chloroplasts and bacteria are comprised of two functionally distinct sectors: Fo, a membraneembedded sector containing hydrophobic proteins that constitute a proton channel, and F1, a soluble aggregate on which adenine nucleotide interconversions are catalyzed [l]. In the fully assembled FoF I-ATPase complex ATP synthesis is coupled to proton translocation. A key component of the proton channel is a dicyclohexylcarbodiimide-binding proteolipid [2], denoted subunit 9 in the mitochondrial ATPase complex (mtATPase) of the yeast Saccharomyces cerevisiae. As with the analogous proteolipids from other FoF,-ATPase complexes [2], the yeast subunit 9 (76 amino acids) [3] has two hydrophobic transmembrane stems separated by a charged hydrophilic loop that is thought to make functional contact with the F1 sector [4]. Aggregates of subunit 9, together with subunit 6 (259 amino acids), are considered to comprise the proton channel in yeast mitochondria [5]. One further Fo component in yeast is subunit 8 (48 amino acids), a proteolipid known to be responsible for the assembly of subunit 6 [5]. In S. cerevisiae, subunits 9, 6 and 8 are specified by the mitochondrial genes olil, oli2 and aapl, respectively [5].

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Section: Synthesis Of Nap9mentioning

confidence: 85%

“…On account of their solubility in chloroform/ methanol [20], both subunit 9 [2] and subunit 8 [25] are termed proteolipids. Fig.…”

Section: Synthesis Of Nap9mentioning

confidence: 99%

Reprogrammed expression of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae

Farrell¹,

Gearing²,

Nagley³

1988

European Journal of Biochemistry

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“…Thus it has been shown that both transmembrane stems (4) and a hydrophilic charged loop (5) of subunit 9 (76 amino acids), together with two of the five or more transmembrane stems (6) of subunit 6 (259 amino acids), participate in protonophoric functions and energy coupling (3). Studies on protein assembly (7) have shown that subunit 8, a 48-amino acid polypeptide (8) with a single transmembrane stem (9), is required for the assembly of subunit 6 into the mtATPase complex, while the assembly of subunit 8 is dependent upon the correct integration of subunit 9 into the membrane (2).…”

mentioning

confidence: 99%

Assembly of functional proton-translocating ATPase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle.

Nagley

Farrell

Gearing

et al. 1988

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

126

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A mitochondrial gene from Saccharomyces cerevisiae encoding a hydrophobic membrane protein, subunit 8 of the FO/Fl-type mitochondrial ATPase complex, has been functionally replaced by an artificial nuclear gene specifying an imported version of this protein. The experiments reported here utilized a multicopy expression vector (pLF1) that replicates in the nucleus of yeast cells and that carries an inserted DNA segment, specifying a precursor protein (N9/Y8) consisting of subunit 8 fused to an N-terminal cleavable transit peptide (the leader sequence from Neurospora crassa ATPase subunit 9). The successful incorporation of the imported subunit 8 into functional ATPase complexes after transformation with pLF1 expressing N9/Y8 was indicated by the efficient genetic complementation of respiratory growth defects of aapl mit-mutants, which lack endogenous subunit 8. The reconstitution of ATPase function was confirmed by biochemical assays of ATPase performance in mitochondria and by immunochemical analyses that demonstrated the assembly of the cytoplasmically synthesized subunit 8 into the ATPase complex. Reconstitution of ATPase function required the cytoplasmically synthesized subunit to have a transit peptide. The strategy for importation and reconstitution developed for subunit 8 leads to a systematic approach to the directed manipulation of mitochondrially encoded membraneassociated proteins that has general implications for exploring membrane biogenesis mechanistically and evolutionarily.Considerable insight into the assembly and function of energy-transducing complexes of microorganisms has been derived from molecular genetic studies (1). The FO/F1-type mitochondrial ATPase (mtATPase) of Saccharomyces cerevisiae is such a complex, in which the three intensely hydrophobic proteins of the FO membrane sector have been subjected to extensive genetic and biochemical analyses (2, 3). The three proteins, subunits 6, 8, and 9, are encoded by yeast mtDNA. A multifaceted approach has been applied, including mutational analysis by gene sequencing and detailed investigation of the mutant phenotypes at physiological and molecular levels. The data obtained have begun to define the roles of individual amino acid residues and domains of these proteins in their interactive assembly into the membrane, in the function of the proton channel of FO, and in energy-coupling leading to ATP synthesis catalyzed by the soluble F1 sector of the complex. Thus it has been shown that both transmembrane stems (4) and a hydrophilic charged loop (5) of subunit 9 (76 amino acids), together with two of the five or more transmembrane stems (6) of subunit 6 (259 amino acids), participate in protonophoric functions and energy coupling (3). Studies on protein assembly (7) have shown that subunit 8, a 48-amino acid polypeptide (8) with a single transmembrane stem (9), is required for the assembly of subunit 6 into the mtATPase complex, while the assembly of subunit 8 is dependent upon the correct integration of subunit 9 into the membrane (2).A...

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“…These proteins may be called proteolipids which are extracted from the samples by using organic solvents such as a mixture of chloroform and methanol (Velours et al, 1984). An aqueous solution containing mild detergents such as Triton X-100 and CHAPS is an alternative way to dissolve the proteins from cells or organelles.…”

Section: Extraction Of Lipid-soluble Proteinmentioning

confidence: 99%

Protein Purification

Sattayasai¹

2012

Chemical Biology

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Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae.

Cited by 70 publications

References 42 publications

Reprogrammed expression of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae

Reprogrammed expression of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae

Assembly of functional proton-translocating ATPase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle.

Protein Purification

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