Abstract
Co-reconstitution of subunits E and G of the yeast V-ATPase and the alpha and beta subunits of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) resulted in an alpha(3)beta(3)EG hybrid complex showing 53% of the ATPase activity of TF(1). The alpha(3)beta(3)EG oligomer was characterized by electron microscopy. By processing 40,000 single particle projections, averaged two-dimensional projections at 1.2-2.4-nm resolution were obtained showing the hybrid complex in various positions. Difference mapping of top and side views of this complex with projections of the atomic model of the alpha(3)beta(3) subcomplex from TF(1) (Shirakihara, Y., Leslie, A. G., Abrahams, J. P., Walker, J. E., Ueda, T., Sekimoto, Y., Kambara, M., Saika, K., Kagawa, Y., and Yoshida, M. (1997) Structure 5, 825-836) demonstrates that a seventh mass is located inside the shaft of the alpha(3)beta(3) barrel and extends out from the hexamer. Furthermore, difference mapping of the alpha(3)beta(3)EG oligomer with projections of the A(3)B(3)E and A(3)B(3)EC subcomplexes of the V(1) from Caloramator fervidus (Chaban, Y., Ubbink-Kok, T., Keegstra, W., Lolkema, J. S., and Boekema, E. J. (2002) EMBO Rep. 3, 982-987) shows that the mass inside the shaft is made up of subunit E, whereby subunit G was assigned to belong at least in part to the density of the protruding stalk. The formation of an active alpha(3)beta(3)EG hybrid complex indicates that the coupling subunit gamma inside the alpha(3)beta(3) oligomer of F(1) can be effectively replaced by subunit E of the V-ATPase. Our results have also demonstrated that the E and gamma subunits are structurally similar, despite the fact that their genes do not show significant homology.
Highlights
Vacuolar-type ATPases (V-ATPases)1 are membrane-bound proteins functioning in active ion pumping at the expense of ATP hydrolysis
The position of the E and G subunits in this hybrid complex was determined after single particle image analysis compared with projections of the ␣33 molecular model of thermophilic F1-ATPase (TF1)- [30] and V1-ATPase subcomplexes from C. fervidus [25]
Reconstitution of an a33EG or a33G Hybrid Complex— Recent work has shown that an ␣33E hybrid complex, consisting of the major ␣ and  subunits of the thermophilic F1-ATPase (TF1) and V-ATPase subunit E (Vma4p) from S. cerevisiae, can be assembled that is functional in ATP hydrolysis [27]
Summary
V-ATPase, vacuolar-type ATPase; FATPase, F1Fo-ATP synthase; TF1, F1-ATPase from Bacillus strain PS3; MRA, multireference alignment. V-ATPases are responsible for acidification of intracellular compartments and generation of an electrochemical gradient (ion motive force) [4, 5] These enzymes consist of two structural and functional parts, a hydrophilic, V1, and hydrophobic, VO, portion. A hydrolytically active ␣33E hybrid complex, composed of the ␣ and  subunits of the thermophilic F-ATPase and subunit E (Vma4p) of the Saccha-. Structure of a F1-/V1-ATPase Hybrid Complex romyces cerevisiae V-ATPase, could be assembled, indicating a major role of subunit E in ATP hydrolysis [27]. We have turned our attention to the first structural determination of a larger ATPase active F1/V1 hybrid complex, ␣33EG This complex contains the V-ATPase subunit G (Vma10p), which has been proposed to be essential for the assembly and activity of the V-ATPase [24, 28, 29]. The position of the E and G subunits in this hybrid complex was determined after single particle image analysis compared with projections of the ␣33 molecular model of TF1- [30] and V1-ATPase subcomplexes from C. fervidus [25]
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