cDNA cloning and sequencing for the import precursor of subunit b in H+-ATP synthase from rat mitochondria

Tsurumi, Chizuko; Yoshihara, Yutaka; Osaka, Fumio; Yamada, F.; Tani, Isamu; Higuti, Tomihiko; Shimizu, Makoto; Oeda, Kenji; Ohkawa, Hiromi; Toda, Hirofumi; Kakuno, Tomisaburo; Sakiyama, Fumio; Kumatori, Atsushi; Tanaka, Keiji; Ichihara, Akira

doi:10.1016/0006-291x(90)91444-w

Cited by 16 publications

(10 citation statements)

References 18 publications

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“…As described previously [20], the probes used were a XhoI± BglII fragment of the cDNA for subunit b [21], an AccI±AvaI fragment of cDNA for subunit d [22], a HindIII±HindIII fragment of cDNA for subunit e [23], a HindIII±ApaLI fragment of cDNA for OSCP [24], a HindIII±ApaLI fragment of cDNA for IF1 [24], a HindIII±HindIII fragment of cDNA for F6 [25], a…”

Section: P-labeled Probe Dnasmentioning

confidence: 99%

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Synchronized transcriptional gene expression of H+-ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Himeda¹,

Morokami²,

Arakaki³

et al. 2000

Eur J Biochem

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Little is known about the relationship between the stoichiometry of polypeptides of multisubunit enzyme complexes and the absolute amount of each transcript of the complexes in mammalian tissues. Here we showed that the absolute amounts of the transcripts of most subunits of rat H 1 -ATP synthase examined greatly differed in the different tissues, showing the following hierarchy of tissue-specificity: heart . kidney . brain < liver. However, surprisingly, there was no difference in the expression pattern of these in terms of the molar ratio of each transcript, indicating a nearly similar stoichiometric expression pattern irrespective of tissue or age of the rat. Therefore, the present finding clearly indicates that most of the transcripts of the 16 subunits of rat H 1 -ATP synthase were concertedly and synchronously expressed, having a constant expression pattern of the transcripts, irrespective of tissue or age of the rats. This is the first report of the absolute amounts of the transcripts of this multisubunit enzyme.Keywords: H 1 -ATP synthase; expression pattern; transcript; stoichiometry; synchronization.The mechanism of the transcriptional regulation of the genes involved in the biosynthesis and assembly of multisubunit mitochondrial enzyme complexes, e.g. [14,17±18a]. Subunit a and A6L are encoded by the mitochondrial genome, and all of the other subunits are separately encoded by nuclear chromosomes; however, the relationship between the stoichiometry of polypeptides of the complexes and the absolute amount of each transcript of this multisubunit enzyme complex in mammalian tissues is still entirely unknown.In the present work, we determined the absolute amount of nine species of mRNAs for eight nucleus-encoded subunits of H 1 -ATP synthase in the brain, liver, heart, and kidney tissues of 2-week-old to 90-week-old male Fischer 344 rats by using highly purified poly(A) 1 RNA and the mRNAs synthesized by an in vitro transcriptional system. M A T E R I A L S A N D M E T H O D S AnimalsMale Fischer 344 rats were obtained from Charles River Japan Inc (Atsugi, Japan), and were genetically identical because of approximately 300 matings between sister and brother. Purification of Poly(A)1 RNAThe rats were decapitated, and tissues of brain, liver, heart, and kidney were rapidly removed. Total RNA from these tissues was isolated by the guanidinium thiocyanate method [19], followed by centrifugation at 120 000 g for 24 h in 5.7 m CsCl solution and then extraction with acid phenol. The poly(A) 1 RNA was purified with Oligotex-dT30 (Takara) according to the manufacturer's instructions, and the purification steps with Oligotex-dT30 were repeated twice. The poly(A) 1 RNA eluted from the 2nd Oligotex-dT30 was extracted with acid phenol, precipitated with 70% ethanol, dried, and dissolved in diethyl pyrocarbonate-treated water. The amount of poly(A) 1 RNA was determined from the absorbance at 260 nm (one unit of absorbance at 260 nm 40 mg´mL 21). The A260/A280 ratios were in the range 1.8±2.0. Contamination by ribosoma...

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Section: P-labeled Probe Dnasmentioning

confidence: 99%

“…2). synthase subunits were synthesized by the MAXIscript In Vitro Transcription Kit as described previously [21]. The obtained synthetic mRNAs were denatured and electrophoresed as described previously [21], and then stained with SYBRw Green I (Wako).…”

Section: R E S U L T Smentioning

confidence: 99%

Synchronized transcriptional gene expression of H+-ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Himeda¹,

Morokami²,

Arakaki³

et al. 2000

Eur J Biochem

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“…The remainder of its sequence is highly charged and is capable of making important interactions either with other subunits in the stalk or, as in the analogous subunit b in the bacterial F,F,-ATPases, with F, subunits. Homologues of bovine b are present in the rat [29] and yeast [30] mitochondrial enzymes, and a human homologue has also been sequenced [31]. Subunit d has no extensive hydrophobic regions in its sequence [28], and, like F,, it is probably attached to the matrix surface of the inner mitochondrial membrane.…”

Section: F1 -Atpasementioning

confidence: 99%

The role of the stalk in the coupling mechanism of F₁F₀‐ATPases

Walker

Collinson

1994

FEBS Letters

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The extrinsic and intrinsic membrane sectors of F,F,-ATPases are linked by a slender stalk 4&50 A in length. The stalk transmits the energy produced by oxidative or photosynthetic phosphorylation from the intrinsic sector, FO, to the catalytic sites in the extrinsic F, sector. How this is achieved is unknown, but long-range conformational changes linked to transmembrane proton transport may be involved. In bacterial and chloroplast F,F,-ATPases, the stalk is probably a composite of subunits 6 and E, part of the y-subunit, and the extrinsic membrane domains of 2 subunits (identical or non-identical according to the species) that are bound to the membrane by their N-terminal regions. The stalk in the bovine mitochondrial enzyme appears to be more complex, and the y, 6, E, OSCP, Fe, b and d subunits all contribute to it. A bovine stalk complex has been assembled in vitro from bacterially expressed OSCP, F,, b and d, both in the presence and in the absence of F,-ATPase. One molecule of each of these subunits is present in the assembled complexes, as there is also in each native F,F,-ATPase assembly. Providing that suitable crystals can be obtained, the stalk complex and the F, stalk complex may permit the high resolution structure of bovine F,-ATPase to be extended into the stalk domain.

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“…1, the probes used were a XhoI-BglII fragment of cDNA for subunit b (13), an AccI-AvaII fragment of cDNA for subunit d (17), a HindIII-HindIII fragment of cDNA for subunit e (15), a HindIII-ApaLI fragment of cDNA for OSCP (16), a HindIII-ApaLI fragment of cDNA for IF1 (16), a HindIII-HindIII fragment of cDNA for F 6 (14), a XhoI-EcoRI fragment of cDNA for subunit c(P1) (16), and an AccI-EcoO1091 fragment of cDNA for subunit c(P2) (16). The probe for ␤-subunit mRNA was a fragment of 634 -925 bp of ␤-subunit cDNA (21) that was synthesized by a nested reverse transcription-polymerase chain reaction method using four primers (outer primers: 5Ј-ATAAGGTTGTGGATCTGCTGGCC-3Ј and 5Ј-AACTCAGCAATAGCACGGGACAGCA-3Ј; inner primers: 5Ј-GGGATATCGCGTTGGTATATGGGCAGATGA-3Ј and 5Ј-GGGCGGC-CGCACATAGATAGCCTGCACTGAG-3Ј).…”

Section: Purification Of Poly(a)mentioning

confidence: 99%

“…We then determined the primary sequences of the synthase subunits by the protein sequence and cDNA cloning techniques (13)(14)(15)(16)(17).…”

mentioning

confidence: 99%

Gene Expression of Subunit c(P1), Subunit c(P2), and Oligomycin Sensitivity-conferring Protein May Play a Key Role in Biogenesis of H+-ATP Synthase in Various Rat Tissues

Sangawa

Himeda

Shibata

et al. 1997

Journal of Biological Chemistry

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cDNA cloning and sequencing for the import precursor of subunit b in H+-ATP synthase from rat mitochondria

Cited by 16 publications

References 18 publications

Synchronized transcriptional gene expression of H+-ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Synchronized transcriptional gene expression of H+-ATP synthase subunits in different tissues of Fischer 344 rats of different ages

The role of the stalk in the coupling mechanism of F₁F₀‐ATPases

Gene Expression of Subunit c(P1), Subunit c(P2), and Oligomycin Sensitivity-conferring Protein May Play a Key Role in Biogenesis of H+-ATP Synthase in Various Rat Tissues

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cDNA cloning and sequencing for the import precursor of subunit b in H+-ATP synthase from rat mitochondria

Cited by 16 publications

References 18 publications

Synchronized transcriptional gene expression of H+-ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Synchronized transcriptional gene expression of H+-ATP synthase subunits in different tissues of Fischer 344 rats of different ages

The role of the stalk in the coupling mechanism of F1F0‐ATPases

Gene Expression of Subunit c(P1), Subunit c(P2), and Oligomycin Sensitivity-conferring Protein May Play a Key Role in Biogenesis of H+-ATP Synthase in Various Rat Tissues

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The role of the stalk in the coupling mechanism of F₁F₀‐ATPases