E. coli Fi-ATPase

E. coli Fi -ATPase consists of the structure aifijydc. The a-subunit and /3-subunit each independently possesses a nucleotide-binding site and the catalytic site is believed to be on the /3-subunit or at the interface between the a- and /3-subunits (for reviews, see Ref. 59 and Chapter 11 of this volume). E. coli Fi -ATPase was more effectively inactivated by AP3-PL and AP4-PL than AP2-PL. The binding of one mole of AP3-PL to one mole of Fi resulted in an almost complete loss of activity 70% of the label bound to Fi to the a-subunit and the rest to the /3-subunit. The sequence analysis of the Fi modified by AP3-PL in the absence of Mg2+ revealed that Lys-201 of the a-subunit and Lys-155 of the /3-subunit are the... 

All five subunits of the E. coli Fi ATPase have been purified, and the primary sequence of the a-subunit has been determined. ATPase activity could be reconstituted from the a-, B- and y-subunits. Subunits j3 and y from R. rubrum have been purified and reincorporated into the depleted membrane with full activity. The a- and /3-subunits contain sites for nucleotides. The sensitivity of the a-subunit of E. coli to trypsin hydrolysis differs when the high affinity site for ATP is saturated, suggestii that binding of ATP produces conformational changes. ... 

J. Weber, S. Wilke-Mounts, R.S. Lee, E. GreU, A.E. Senior, Specific placement of tryptophan in the catalytic sites of the Escherichia coli Fi-ATPase provides a direct probe of nucleotide binding maximal ATP hydrolysis occurs with three sites occupied. J. Biol. Chem. 268, 20126-20133 (1993)... 

A number of active -subunits have been isolated from various Fi-ATPases after their complete dissociation into individual subunits (1-3). Two of these isolated / -subunits, the E, coli Fi P subunit(Ec 3) and the chloroplast CFi-/ subunit (CFi/ ), were shown to form hybrid FqFi-complexes upon their reconstitution into / -less R, rubrum chromatophores (3,4). These hybrid complexes restored a high Mg-ATPase activity, but very little ATP synthesis, in the inactive )5-less chromatophores. On the other hand, reconstitution of the native R, rubrum Fi-/3 subunit (Rr/3), that has been removed from the chromatophore membrane-bound FoFi-ATP synthase by extraction with LiCl (5,6) led to full restoration of both coupled ATP synthesis and hydrolysis by the / -less chromatophores. 

Fig. 37. Interaction of residues ofthe e-subunit of EcFi-ATPase with the a- and p-subunits of Fo Fi-ATPase known from cross-linking studies and modeled by the bovine MFi-ATPase structure. Figure source Uhlin, Cox and Guss (1997) Crystal structure ofthe e subunit of the proton-translocating ATP synthase from Escherichia coli. Structure 5 1227.

In the FoFi-ATPase of mammalian and yeast mitochondria, the oligomy-cin sensitivity conferring protein (OSCP) is one of the subunits of the enzyme complex, and is needed for inhibition of ATP hydrolysis by oligomycin (1). The amino acid sequence of OSCP from beef heart mitochondria is homologous to the amino acid sequence of the S-subunit of the Fi-ATPase from E. coli, 26.4% (2), chloroplasts, 25.3% (3), and Rhodospirill urn rubrum, 28.9% (2). However, only R. rubrum is sensitive to oligomycin (4). It has also been shown that R. rubrum Fj-ATPase, with the B-subunits substituted with B from E. coli, is not oligomycin sensitive when reconstituted to depleted membranes (5). 

Abbreviations used Fi Fq, H+-translocating ATP synthases from mitochondria, E. coli plasma membranes, plasma membranes of the thermophilic bacterium PS3 and chloroplast membranes CFi, chloroplast coupling factor one ATPase, CFiE. C. 3.6.1.3 SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl... 

The evolutionary relations among subunits of F from several sources were tested by immunological cross-reactivity. Antibodies raised against subunits of Fi were reacted with H -ATPase complex from Chlamydomonas and spinach chloroplasts, from beef adrenal, rat liver and yeast mitochondria and with E. coli membranes. The antigen-antibody reaction was tested employing the electrotransfer technique (Rott, Nelson 1981 Towbin et al. 1979). The antibodies employed were raised against the following subunits of the H -ATPase complex o(,, < and... 

Walker JE, Runswick MJ and Saraste M (1982) Subunit equivalence in E.coli and bovine heart mitochondrial Fi Fo ATPases, FEBS Lett. 146, 393-396. 

The Mg2+-activated ATPase (or ATP synthase) is made up of two parts. The Fj component is the catalytic, Mg2+-binding, extrinsic membrane protein composed of five different subunits, a, (3, y, S and e. The F0 component is an intrinsic membrane complex that contains three subunits, a, b and c, and mediates proton translocation. The F, protein is bound to the membrane through interaction with F0. The complexity of the F,F0 enzyme has presented many difficulties. Hie greatest advances have been made for the bacterial enzymes, notably for thermophiles, Escherichia coli and Rhodospirillum rubrum, where progress has been made in the purification of subunits and their reconstitution into membranes, and the identification of binding sites for Mg2+ and nucleotides on the Fi subunits.300 FiF0 preparations can be incorporated into liposomes and display H+ translocation, ATP-P, exchange and ATP synthesis.301... 

---