Evidence for Major Structural Changes in the Manduca sexta Midgut V1 ATPase Due to Redox Modulation

Abstract

The shape and overall dimensions of the oxidized and reduced form of the V 1 ATPase from Manduca sexta were investigated by synchrotron radiation x-ray solution scattering. The radius of gyration of the oxidized and reduced complex differ noticeably, with dimensions of 6.20 ؎ 0.06 and 5.84 ؎ 0.06 nm, respectively, whereas the maximum dimensions remain constant at 22.0 ؎ 0.1 nm. Comparison of the low resolution shapes of both forms, determined ab initio, indicates that the main structural alteration occurs in the head piece, where the major subunits A and B are located, and at the bottom of the stalk. In conjunction with the solution scattering data, decreased susceptibility to tryptic digestion and tryptophan fluorescence of the reduced V 1 molecule provide the first strong evidence for major structural changes in the V 1 ATPase because of redox modulation.Ion-translocating, vacuolar-type, ATPases (V-ATPases), which use ATP to acidify intracellular compartments and energize plasma membranes, consist of a hydrophilic, catalytic complex, V 1 , and a hydrophobic, ion-translocating channel complex, V O (1, 2). In the larval midgut of the tobacco hornworm, Manduca sexta, the V O complex is made up of four subunits with apparent molecular masses of 100 (a), 40 (d), 20 (e) and 17 (c) kDa. The hydrophilic portion, V 1 ATPase, consists of eight distinct subunits of molecular mass 67 (A), 56 (B), 54 (H), 40 (C), 32 (D), 28 (E), 16 (G), and 14 kDa (F). Three interdigitating copies each of the nucleotide-binding subunits A and B form a head piece at the top of the molecule, whereas the remaining V 1 subunits form a stalk connecting the head piece to the V O complex (3).Several mechanisms have been proposed for the regulation of V-ATPases, including disassembly and reassembly of peripheral V 1 and integral V O complexes (4 -7), regulation of parallel ion channels that compensate for the electrogenicity of the V-ATPase (8, 9), changes in the degree of coupling between ATP hydrolysis and proton pumping (9, 10), and reversible disulfide bond interchange between conserved cysteine residues near the catalytic site (reviewed in Ref. 11). Studies on the bovine clathrin-coated V-ATPase have shown that disulfide bond formation between the conserved residues Cys 254 and Cys 532 of the catalytic A subunit leads to reversible inactivation of the enzyme (12, 13). Such a reversibly inactivated, disulfidebonded state has been found in significant fractions of V-ATPase in native clathrin-coated vesicles (12). Disulfide bonds at the catalytic site of the V-ATPase are also induced by the nitric oxide-generating reagent S-nitrosoglutathione (14). This is consistent with the finding that in Neurospora crassa oxidizing and reducing agents have inhibitory and stabilizing effects, respectively, on the enzyme (15). Moreover, a mutation in the cysteine biosynthetic pathway in yeast leads to defective vacuolar acidification, which can be corrected by a C261V mutation, suggesting that disulfide bond formation can cause inhibition of V-ATPase activity ...

Subunit Arrangement in V-ATPase from Thermus thermophilus

“…Cross-linking studies have suggested that the D subunit was adjacent to B subunit at central cavity region of A 3 B 3 hexamer, and the F subunit was associated with the D subunit (29,30). In contrast, studies on the V-ATPase from Manduca sexta suggested that subunit E, rather than subunit D, was the rotor subunit (31). Electron microscopic study of Na ϩ -pumping V 0 V 1 -ATPase from Caloramator fervidus also suggested that the E subunit was the rotor (32).…”

“…The V 0 part can be divided into two complexes, one is composed of subunits E, G, and I, and the other is composed of subunits L and C. The E subunit was predicted to be a highly hydrophilic ␣-helical protein and one of candidates of F 1 -␥ subunit homologue (31,32). Our results are consistent with the cross-linking studies by Arata et al (29,30) and strongly suggest that the subunit E is a stator subunit, rather than a rotor subunit.…”

Resolution of the V1 ATPase from Manduca sexta into Subcomplexes and Visualization of an ATPase-active A3B3EG Complex by Electron Microscopy

“…Several lines of evidence indicate that redox-modulation of the V-ATPase, proposed as a mechanism of regulation of this complex (29 -31), leads to structural changes in the enzyme (3,29,32). In this connection we incubated V 1 ATPase with 0.1% LDAO in the absence of the reducing agent, 2-mercaptoethanol.…”

“…The two major subunits A and B, in a stoichiometry of A 3 :B 3 , contain the nucleotide-binding sites and are connected to the V O part by the so-called stalk subunits C-H (6). Seen from the side the structures of the V 1 ATPase, recently identified from Caloramator fervidus (7) and the tobacco hornworm, M. sexta (8,9), revealed a molecule with a single, compact stalk.The V 1 ATPase from M. sexta, which reversibly dissociates from the V O part as an in vivo regulatory mechanism (10), is the object of our studies and comprises the eight subunits A, B, H, C, D, E, G, and F with apparent molecular masses of 67, 56, 54,40,32,28,14, and 16 kDa, respectively (11). Low resolution structural studies of this V 1 complex using small-angle x-ray scattering have shown that the hydrated enzyme is an elongated molecule.…”

“…The V 1 ATPase from M. sexta, which reversibly dissociates from the V O part as an in vivo regulatory mechanism (10), is the object of our studies and comprises the eight subunits A, B, H, C, D, E, G, and F with apparent molecular masses of 67, 56, 54,40,32,28,14, and 16 kDa, respectively (11). Low resolution structural studies of this V 1 complex using small-angle x-ray scattering have shown that the hydrated enzyme is an elongated molecule.…”