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Mechanisms of Bioenergetic Membrane Proteins Structural features and nucleotide-binding capability of the C subunit are integral to the regulation of the eukaryotic V1Vo ATPases G. Grüber1 Universität des Saarlandes, Fachrichtung 2.5-Biophysik, Universitätsbau 76, D-66421 Homburg, Germany Abstract V-ATPases (vacuolar ATPases) are responsible for acidification of intracellular compartments and, in certain cases, proton transport across the plasma membrane of eukaryotic cells. They are composed of a catalytic V1 sector, in which ATP hydrolysis takes place, and the Vo sector, which functions in proton conduction. The best established mechanism for regulating the V-ATPase activity in vivo involves reversible dissociation of the V1 and Vo domains, in which subunit C is intimately involved. In the last year, impressive progress has been made in elucidating the structure of the C subunit and its arrangement inside the V-ATPase. Nucleotide occupancy by subunit C, followed by conformational changes of this subunit has shed light on the mechanism of V-ATPase regulation. Introduction V-ATPase (vacuolar ATPase) is an electrogenic ion pump found in every eukaryotic cell. This enzyme harnesses the energy derived from ATP hydrolysis to pump ions across membranes, thereby creating an electrochemical gradient. V-ATPases have two structural and functional parts, a peripheral V1 complex, whose catalytic part faces the cytosol, and a membrane-bound ion-conducting Vo part. The eukaryotic enzyme V1 consists of eight subunits A–H, whereas the Vo domain is composed of the different subunits a, c, c , c , d and e [1]. ATP is hydrolysed on the V1 -headpiece, composed of an A3 B3 hexamer, and the energy released during that process is transmitted to the membrane-bound Vo domain, to drive ion translocation. This energy coupling occurs via the so-called ‘stalk’ structure, an assembly of the V1 and Vo subunits C–H and a respectively that forms the functional and structural interface [2]. A characteristic feature of the eukaryotic V1 Vo ATPase is the regulation by reversible disassembly of the V1 and Vo subcomplexes [3–5], resulting in the decrease of Mg2+ dependent ATPase activity and proton pumping across the membrane. Reassembly of both domains restores these activities. It was shown that subunits C and H are important for inhibition of the Mg2+ -dependent ATPase activity of dissociated V1 complexes [4]. The high-resolution structure of the H subunit [6] and data on the gross structure of the V1 Vo ATPase complexes suggest that, in the intact enzyme, this subunit is involved in the formation of the peripheral stalk region [7], despite the fact that a rearrangement within the Key words: reversible dissociation, vacuolar-type ATPase, V1 Vo ATPase, Vma5p nucleotidebinding. Abbreviations used: EM, electron microscopy; SAXS, small-angle X-ray scattering; V-ATPase, vacuolar ATPase. 1 Present address: School of Biological Sciences, Nanyang Technological University, Singapore 637551 (email [email protected]). disassembled V1 is possible [2]. Electron microscopy studies of the disassembled V1 complex from tobacco hornworm Manduca sexta have shown that subunit C dissociates from the V1 subcomplex [8], although it is essential for the reassembly of the functional V1 Vo [3,4]. Recent work on the structure of subunit C, the relationship of nucleotide binding of C and interactions of this stalk subunit within the V1 ATPase is reviewed here. Quaternary structure of the V1 ATPase The fundamental aim of structural studies in molecular biology is to establish a relationship between structure (or, more precisely, structural changes) and function of biological molecules. Over the past years, a significant amount of structural information about the V1 ATPase has been obtained using three-dimensional EM (electron microscopy) [8,9], SAXS (small-angle X-ray scattering) [10,11] and threedimensional crystallography [6,12]. Significant insights into the molecular mechanism of ATP hydrolysis came from the three-dimensional structure of the V1 ATPase without subunit C [V1 (−C)] from M. sexta at 18 Å (1 Å = 0.1 nm) resolution [8] (Figure 1), providing a picture of where the three A and B subunits form a hexagon, alternating around a central cavity, in which a seventh mass is located. This seventh mass is not located in the centre of the cavity of the A3 B3 hexamer, but is slightly offset to one side, thereby strengthening the interaction between two non-neighbouring A subunits [8]. This feature is comparable with the asymmetric location of the rotating γ subunit in the α 3 β 3 subcomplex of the related F-ATPase [13], indicating significant movements of the central mass in V-ATPases during catalysis. In a side view, the structure shows three protuberances at the top of the A3 B3 headpiece, which might belong to the N-termini of subunit A [8]. At the bottom side C 2005 Biochemical Society 883 884 Biochemical Society Transactions (2005) Volume 33, part 4 Figure 1 The 18 Å structure of the V1 ATPase without subunit C Figure 2 Low-resolution structure of subunit C of the V-ATPase from M. sexta Modified with permission from M. Radermacher, T. Ruiz, H. Wieczorek from S. cerevisiae derived from SAXS data [11]. and G. Grüber (2001) The structure of the V1 -ATPase determined by three-dimensional electron microscopy of single particles. J. Struct. Biol. c Elsevier. 135, 26–37 of the A3 B3 domain, the stalk protrudes with an angle of approx. 7◦ with the vertical axis of the cavity. The length of the stalk in the V1 (−C) complex is shorter than the value determined for the complete and hydrated V1 ATPase (11 nm) using SAXS [10]. This difference has been caused by the separation of the C subunit in the V1 (−C) complex used in the (EM) studies [8]. Structural features and arrangement of subunit C and H Previously, the structure of the C subunit (Vma5p) from the yeast V1 Vo ATPase has been studied by SAXS [11]. The radius of gyration Rg and the maximum dimension Dmax of subunit C are 3.74 ± 0.03 nm and 12.5 ± 0.1 nm respectively, suggesting that the subunit is a rather elongated particle. The gross structure of subunit C was restored ab initio from the scattering data, revealing that the hydrated Vma5p has an elongated boot-shaped feature [11] (Figure 2). A recent 1.75 Å map from X-ray diffraction studies of subunit C (Vma5p, [12]) confirms this feature and shows that this subunit consists of three distinct domains. An upper head domain, composed of amino acids 166–263, a large globular foot, consisting of the N- and C-termini, and an elongated neck domain, which connects the head and foot region. The overall length of the stalk and structure of subunit C indicate that this subunit might span the full stalk, thereby linking the catalytic A3 B3 domain via its head region to the Vo domain via the foot C 2005 Biochemical Society region. This foot region of Vma5p with approx. 5 nm in width and 4.5 nm in height, would fit to the bottom of the stalk domain of the hydrated V1 ATPase, determined by SAXS [10]. These data are in agreement with two-dimensional projections of a hydrolytic active V1 –Vma5p hybrid complex, composed of subunit C (Vma5p) of Saccharomyces cerevisiae V-ATPase and the C-depleted V1 from M. sexta, determined from single particle electron microscopy [14]. The total length of the stalk element in this hybrid complex is approx. 10.5 nm, which exceeds the length of the central stalk in V1 complex lacking subunit C by 4.5 nm. The EM data also suggest that, within the hybrid complex, Vma5p is most likely to be arranged with its long axis parallel to the stalk direction, as shown in Figure 2. Electrostatic analysis also indicates that the foot region of subunit C is oriented to the membrane domain [12]. The boot-shaped structure of subunit C (Vma5p) is remarkably similar in its overall structure to that of subunit H (Vma13p), composed of an elongated bootleg, made up by the N-terminal domain, and a foot, which is formed by the C-terminal domain. The crystal structure of subunit H shows that both domains are connected by a flexible linker [6]. The C-terminal half of subunit H is proposed to bind to the V-ATPase subunits, whereby the long terminal shaft interacts with proteins like Ynd1p or the Nef-binding protein-1 [6]. In comparison, the C-terminus of subunit C (Vma5p), which includes the most highly conserved region of this subunit, appears to be very important for the stable assembly of V1 Vo . Mutagenesis of this region reduced ATPase activity in vitro, because of loss of V1 subunits [4], indicating that subunit C might also be in contact with the V1 subunits via its Cterminal domain (see below). In line with this is the fact that binding of different maleimides to the single cysteine residue (Cys340 ) of the C-terminal region of subunit C prevented binding of this subunit to the V1 (−C) complex [14]. Since binding of different maleimides to Vma5p prevents this subunit from interaction with C-depleted V1 , it has been proposed that the point of the foot, in which Cys340 is located, Mechanisms of Bioenergetic Membrane Proteins forms at least partially the surface for binding to the stalk region of V1 . Nucleotide binding and structural alterations of subunit C Regulation of V-ATPase function in response to physiological stimuli is thought to be a multilevel process. It includes control of the expression of V-ATPase subunit genes, intracellular targeting and translocation from vesicles to the plasma membrane, and reversible dissociation of the Vo and V1 domains, entailing inactivation of the pump and decrease of MgATPase activity (reviewed in [15]). In S. cerevisiae, it has been shown that glucose deprivation induces disassembly of the V1 and Vo section and that subunit C (Vma5p) reversibly leaves the enzyme after removal of glucose, causing the catalytic V1 to detach from the Vo section [4]. As yet, the nature of this signal is not clear. It has been hypothesized that subunit C might act as a sensor for the cellular ADP:ATP level [16]. As observed independently by photoaffinity labelling and fluorescence correlation spectroscopy, subunit C is capable of binding ADP and ATP, whereby ATP binding is weaker than that of ADP [16]. The C-terminal region of subunit C has been mapped to be the nucleotide-binding site. Tryptophan fluorescence quenching and decreased susceptibility to tryptic digestion of subunit C after binding of different nucleotides provides evidence for structural changes in this subunit because of nucleotide binding [16]. It is of particular interest that the V1 Vo disassembly is accompanied by the dissociation of subunit C from the complex [3], and it has been hypothesized that subunit C plays a central role in the reversible reassembly of both domains [3,4] by binding as an anchor protein to the actin-based cytoskeleton and controlling the linkage of the cytoplasmic V1 complex with the actin filaments [3]. One of the proposed actin-binding sites of the protein is located in the C-terminal domain of subunit C [12]. Therefore binding of the nucleotides ADP and ATP to the C-terminus may induce structural changes in the foot region and thereby alter the interaction with other V1 and Vo subunits, and actin. In summary, subunit C is bound to the catalytic domain of V1 via the head domain and the foot region of C is oriented to the membrane portion [12,14]. The elongated feature of subunit C supports its role as a mediator, which facilitates the linkage of V1 and Vo and thereby permits alterations in the V1 Vo ATPase due to nucleotide binding. Subunit C in V1 Vo acts not only as a stabilizer and regulator, but also as a sensor of the cytosolic ATP/ADP ratio [16]. In addition, the significant length of the stalk of V1 , which separates the catalytic part from the ion-conducting part, requires conformational coupling of the stalk subunits during ATP hydrolysis. In contrast with the related F-ATP synthases, in which the rotary stalk subunits γ and ε bind directly to the rotor ring in the Fo domain [17], subunits C of the V-ATPase makes up an adapter which links catalytic site events in V1 with proton conduction in Vo , thereby implying a different coupling mechanism to that described for F-ATP synthases. References 1 Nelson, N. and Harvey, W.R. (1999) Physiol. Rev. 79, 361–385 2 Müller, V. and Grüber, G. (2003) Cell. Mol. 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(2004) EMBO Rep. 5, 1148–1152 13 Abrahams, J.P., Leslie, A.G.W., Lutter, R. and Walker, J.E. (1994) Nature (London) 370, 621–628 14 Chaban, Y, Juliano, S., Boekema, E.J. and Grüber, G. (2004) Biochim. Biophys. Acta 1708, 196–200 15 Nishi, T. and Forgac, M. (2002) Nat. Rev. Mol. Cell Biol. 3, 94–103 16 Armbrüster, A., Hohn, C., Hermesdorf, A., Schumacher, K., Börsch, M. and Grüber, G. (2005) FEBS Lett. 579, 1961–1967 17 Stock, D., Gibbons, C., Arechaga, I., Leslie, A.G. and Walker, J.E. (2000) Curr. Opin. Struct. Biol. 10, 672–679 Received 23 May 2005 C 2005 Biochemical Society 885