ATPase Activity of a Highly Stable α3β3γ Subcomplex of Thermophilic F1 Can Be Regulated by the Introduced Regulatory Region of γ Subunit of Chloroplast F1

Dirk Bald,Hiroyuki Noji,Michael T Stumpp,Masasuke Yoshida,Toru Hisabori

doi:10.1074/jbc.275.17.12757

Dirk Bald, Hiroyuki Noji + Show 3 more

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https://doi.org/10.1074/jbc.275.17.12757

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Abstract

A mutant F(1)-ATPase alpha(3)beta(3)gamma subcomplex from the thermophilic Bacillus PS3 was constructed, in which 111 amino acid residues (Val(92) to Phe(202)) from the central region of the gamma subunit were replaced by the 148 amino acid residues of the homologous region from spinach chloroplast F(1)-ATPase gamma subunit, including the regulatory stretch, and were designated as alpha(3)beta(3)gamma((TCT)) (Thermophilic-Chloroplast-Thermophilic). By the insertion of this regulatory region into the gamma subunit of thermophilic F(1), we could confer the thiol modulation property to the thermophilic alpha(3)beta(3)gamma subcomplex. The overexpressed alpha(3)beta(3)gamma((TCT)) was easily purified in large scale, and the ATP hydrolyzing activity of the obtained complex was shown to increase up to 3-fold upon treatment with chloroplast thioredoxin-f and dithiothreitol. No loss of thermostability compared with the wild type subcomplex was found, and activation by dithiothreitol was functional at temperatures up to 80 degrees C. alpha(3)beta(3)gamma((TCT)) was inhibited by the epsilon subunit from chloroplast F(1)-ATPase but not by the one from the thermophilic F(1)-ATPase, indicating that the introduced amino acid residues from chloroplast F(1)-gamma subunit are important for functional interaction with the epsilon subunit.

Highlights

A mutant F1-ATPase ␣3␤3␥ subcomplex from the thermophilic Bacillus PS3 was constructed, in which 111 amino acid residues (Val92 to Phe202) from the central region of the ␥ subunit were replaced by the 148 amino acid residues of the homologous region from spinach chloroplast F1-ATPase ␥ subunit, including the regulatory stretch, and were designated as ␣3␤3␥(TCT) (Thermophilic-Chloroplast-Thermophilic)
No loss of thermostability compared with the wild type subcomplex was found, and activation by dithiothreitol was functional at temperatures up to 80 °C. ␣3␤3␥(TCT) was inhibited by the ⑀ subunit from chloroplast F1-ATPase but not by the one from the thermophilic F1-ATPase, indicating that the introduced amino acid residues from chloroplast F1-␥ subunit are important for functional interaction with the ⑀ subunit
An expression plasmid for an ␣3␤3␥ subcomplex of TF1 containing a TF1/chloroplast F0F1 and F1 (CF1) chimeric ␥ subunit (␣3␤3␥(TCT)) was constructed, and the desired protein complex was successfully overexpressed and purified with a yield comparable to those of wild type ␣3␤3␥ [7] or ␣3␤3␥ (␤-His, ␥S106C) [14]. This proves that combining two halves of ␥ subunits from different organisms and the insertion of additional amino acid residues from the ␥ subunit of CF1 into the central part of the ␥ subunit of TF1 are no major obstacles for biosynthesis and in vivo stability of this protein complex in the employed expression system

Summary

Introduction

A mutant F1-ATPase ␣3␤3␥ subcomplex from the thermophilic Bacillus PS3 was constructed, in which 111 amino acid residues (Val to Phe202) from the central region of the ␥ subunit were replaced by the 148 amino acid residues of the homologous region from spinach chloroplast F1-ATPase ␥ subunit, including the regulatory stretch, and were designated as ␣3␤3␥(TCT) (Thermophilic-Chloroplast-Thermophilic). The resulting chimeric ␣3␤3␥ complex, which had substantial ATPase activity, was clearly regulated by the disulfide/dithiol state of the two regulatory cysteine residues. We could demonstrate the importance of the region around the disulfide bridge of ␥ subunit for the regulatory interaction with ⑀ subunit which is known to inhibit activity [28]. Stability of this chimeric complex was still lower than that of wild type thermophilic ␣3␤3␥, and large scale preparation was difficult. In order to understand the molecular mechanism of thiol regulation, we engineered a chimeric ␣3␤3␥ subcomplex of TF1 in which the central half of the ␥ subunit was replaced by the.

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