Interaction of the chloroplast ATP synthetase with the photoreactive nucleotide 3′‐<i>O</i>‐(4‐benzoyl)benzoyl adenosine 5'‐diphosphate

Bar-Zvi, Dudy; Tiefert, Marjorie A.; Shavit, Noun

doi:10.1016/0014-5793(83)80973-9

Cited by 32 publications

(9 citation statements)

References 26 publications

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“…4) might be explained by different accessibility of tentoxin to its binding site. It is known that the conformation of isolated CF 1 differs from the conformation of the CF 1 sector in the membrane-bound CF 0 CF 1 complex (26,27). On the other hand, photophosphorylation of the mutant enzyme produced by Avni et al (7) containing a tobacco/C.…”

Section: Discussionmentioning

confidence: 99%

Catalytic Properties and Sensitivity to Tentoxin of Chlamydomonas reinhardtii ATP Synthases Changed in Codon 83 of atpB by Site-directed Mutagenesis

Fiedler

Golan

et al. 1997

Journal of Biological Chemistry

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The participation of the amino acid ␤83 in determining the sensitivity of chloroplast ATP synthases to tentoxin was reported previously. We have changed codon 83 of the Chlamydomonas reinhardtii atpB gene by sitedirected mutagenesis to further examine the role of this amino acid in the response of the ATP synthase to tentoxin and in the mechanism of ATP synthesis and hydrolysis. Amino acid ␤83 was changed from Glu to Asp (␤E83D) and to Lys (␤E83K), and the highly conserved tetrapeptide ␤T82-E83-G84-L85 (⌬TEGL) was deleted. Mutant strains were produced by particle gun transformation of atpB deletion mutants cw15⌬atpB and FUD50 with the mutated atpB genes. The transformants containing the ␤E83D and ␤E83K mutant genes grew well photoautotrophically. The ⌬TEGL transformant did not grow photoautotrophically, and no CF 1 subunits were detected by immunostaining of Western blots using CF 1 specific antibodies. The rates of ATP synthesis at clamped ⌬pH with thylakoids isolated from cw15 and the two mutants, ␤E83D and ␤E83K, were similar. However, only the phosphorylation activity of the mutant ␤E83D was inhibited by tentoxin with 50% inhibition attained at 4 M. These results confirm that amino acid ␤83 is critical in determining the response of ATP synthase to tentoxin. The rates of the latent Mg-ATPase activity of the CF 1 s isolated from cw15, ␤E83D, and ␤E83K were similar and could be enhanced by heat, alcohols, and octylglucoside. As in the case of the membrane-bound enzyme, only CF 1 from the ␤E83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the ␤E83K-CF 1 than that of CF 1 from the other two strains. Moreover, the optimal activity of the ␤E83K-CF 1 was also lower. These results suggest that introduction of an amino acid with a positively charged side chain in position 83 in the "crown" domain affects the active conformation of the CF 1 -ATPase.The eukaryotic unicellular green algae Chlamydomonas reinhardtii constitutes a powerful experimental model system for the study of the photosynthetic machinery. It is accessible to genetic analysis and grows photoautotrophically on minimal medium or heterotrophically with acetate as the sole carbon source. These properties have been used to isolate numerous photosynthetic mutants which have helped to examine the function of the photosynthetic apparatus (1).The chloroplast of C. reinhardtii contains approximately 80 copies of its 196-kb 1 circular genome (2). Due to recent progress in the molecular genetics of C. reinhardtii, chloroplast proteins can be altered by site-directed mutagenesis of the corresponding genes followed by transformation into the chloroplast (3-7). Chloroplast transformation was first demonstrated in 1988 by Boynton and co-workers (3), by complementation of an atpB deletion mutant with the cloned wild type gene. The transforming DNA integrates into the recipient chloroplast DNA by homologous recombination. Goldschmidt-Clermont (5) has constructed a chimeric selectable marker using ...

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Section: Discussionmentioning

confidence: 99%

Catalytic Properties and Sensitivity to Tentoxin of Chlamydomonas reinhardtii ATP Synthases Changed in Codon 83 of atpB by Site-directed Mutagenesis

Fiedler

Golan

et al. 1997

Journal of Biological Chemistry

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“…Earlier studies using arylazido derivatives of ADP and ATP resulted in the location of the MgATP at a site on or near the (3polypeptide, and site 3 near an interface between both a and f8 subunits (7). Recently, the location of a site on membrane-bound CF1 containing tightly bound ADP has been identified as the P polypeptide with 2-azidoadenosine diphosphate as the label (10) and as both the a and , polypeptides with BzATP as the label (11). In our experiments, however, use of the BzATP substrate analog has resulted in the labeling of all three well-characterized nucleotide binding sites on soluble purified CF1.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, however, photoreactive labeling with 2-azidoadenosine diphosphate bound to membrane-bound CF1 at a site normally containing ADP has resulted in labeling of the 3 polypeptide (10). A similar experiment with 3'-O-(4-benzoyl)benzoyl-ADP resulted in the labeling of both the a and 83 polypeptide chains (11).…”

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confidence: 99%

Investigation of nucleotide binding sites on chloroplast coupling factor 1 with 3'O-(4-benzoyl)benzoyl adenosine 5'-triphosphate.

Kambouris¹,

Hammes²

1985

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

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The subunit locations of each of the three nucleotide binding sites of soluble chloroplast coupling factor 1 have been studied with the photoaffinity label 3'-O-(4-benzoyl)benzoyl-ATP. This derivative is an effective inhibitor of ATPase activity. Photolysis of the radioactive label when bound to each of the three nucleotide sites on the coupling factor has been examined. For the nucleotide site that normally binds ADP very tightly, NaDodSO4/polyacrylamide gel electrophoresis after photolysis indicates that primarily the beta polypeptide chain is appreciably labeled (86%), although some labeling of the alpha polypeptide chain is found (14%). For the site that binds MgATP tightly, 97% of the radioactivity is found on the beta polypeptide chain. The alpha and beta polypeptide chains are labeled in approximately equal amounts when photolysis is carried out with the nucleotide analog bound to the third site.

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“…The compound was labeled with 32Pi using the NDP-Pi exchange reaction catalyzed by polynucleotide phosphorylase. Bz-ADP was synthesized, using either p-32P]ADP (prepared from ADP and 32Pi by NDP-Pi exchange as above) or [8-14C]ADP [7].…”

Section: Methodsmentioning

confidence: 99%

“…Using 2-N3-ADP, it has been observed that both tightly bound ADP and newly synthesized, tightly bound ATP are located on the &subunit of the thylakoid-bound enzyme [5,6]. Peptide mapping of the photolabeled P-subunits demonstrated that the same polypeptide regions of the b-subunit interact with the base portions of both the tightly bound ADP and ATP [7]. In contrast, the LY-and P-subunits of the thylakoid-bound CFt are both labeled by tightly bound Bz-ADP [8].…”

Section: Abbreviationsmentioning

confidence: 99%

Characterization of nucleotide‐binding sites on the chloroplast coupling factor 1 using two photolabile analogs

et al. 1986

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Nucleotide‐binding sites on the chloroplast coupling factor 1 (CF1) have been probed using two photoreactive ADP analogs: 2‐azido‐ADP (2‐N3‐ADP) and 2',3'‐O‐(4‐benzoyl)benzoyl‐ADP (Bz‐ADP). Photolabeling of the isolated CF1 with 2‐N3‐ADP results in incorporation of the analog exclusively into the β‐subunit of the enzyme. The location of the nucleotide‐binding site(s) within the β‐subunit of the CF1 was investigated using peptide mapping. Within the discrimination limits of this technique, it is concluded that the azidoand benzoyl‐modified analogs both bind to the same conformation of the nucleotide‐binding site(s) of soluble CF1. Bz‐ADP, however, labels the binding site(s) on membrane‐bound CF1 in a slightly different manner.

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Interaction of the chloroplast ATP synthetase with the photoreactive nucleotide 3′‐O‐(4‐benzoyl)benzoyl adenosine 5'‐diphosphate

Cited by 32 publications

References 26 publications

Catalytic Properties and Sensitivity to Tentoxin of Chlamydomonas reinhardtii ATP Synthases Changed in Codon 83 of atpB by Site-directed Mutagenesis

Catalytic Properties and Sensitivity to Tentoxin of Chlamydomonas reinhardtii ATP Synthases Changed in Codon 83 of atpB by Site-directed Mutagenesis

Investigation of nucleotide binding sites on chloroplast coupling factor 1 with 3'O-(4-benzoyl)benzoyl adenosine 5'-triphosphate.

Characterization of nucleotide‐binding sites on the chloroplast coupling factor 1 using two photolabile analogs

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