Daniel J. Cipriano

Function, structure and regulation of the vacuolar (H+)-ATPases

The vacuolar ATPases (or V-ATPases) are ATP-driven proton pumps that function to both acidify intracellular compartments and to transport protons across the plasma membrane. Intracellular V-ATPases function in such normal cellular processes as receptor-mediated endocytosis, intracellular membrane traffic, prohormone processing, protein degradation and neurotransmitter uptake, as well as in disease processes, including infection by influenza and other viruses and killing of cells by anthrax and diphtheria toxin. Plasma membrane V-ATPases are important in such physiological processes as urinary acidification, bone resorption and sperm maturation as well as in human diseases, including osteopetrosis, renal tubular acidosis and tumor metastasis. V-ATPases are large multi-subunit complexes composed of a peripheral domain (V(1)) responsible for hydrolysis of ATP and an integral domain (V(0)) that carries out proton transport. Proton transport is coupled to ATP hydrolysis by a rotary mechanism. V-ATPase activity is regulated in vivo using a number of mechanisms, including reversible dissociation of the V(1) and V(0) domains, changes in coupling efficiency of proton transport and ATP hydrolysis and changes in pump density through reversible fusion of V-ATPase containing vesicles. V-ATPases are emerging as potential drug targets in treating a number of human diseases including osteoporosis and cancer.

36° step size of proton-driven c-ring rotation in FoF1-ATP synthase

Synthesis of adenosine triphosphate ATP, the ‘biological energy currency’, is accomplished by FoF1‐ATP synthase. In the plasma membrane of Escherichia coli, proton‐driven rotation of a ring of 10 c subunits in the Fo motor powers catalysis in the F1 motor. Although F1 uses 120° stepping during ATP synthesis, models of Fo predict either an incremental rotation of c subunits in 36° steps or larger step sizes comprising several fast substeps. Using single‐molecule fluorescence resonance energy transfer, we provide the first experimental determination of a 36° sequential stepping mode of the c‐ring during ATP synthesis.

Stochastic high-speed rotation of Escherichia coli ATP synthase F1 sector: The subunit-sensitive rotation

The γ subunit of the ATP synthase F1 sector rotates at the center of the α3β3 hexamer during ATP hydrolysis. A gold bead (40–200 nm diameter) was attached to the γ subunit of Escherichia coli F1, and then its ATP hydrolysis-dependent rotation was studied. The rotation speeds were variable, showing stochastic fluctuation. The high-speed rates of 40- and 60-nm beads were essentially similar: 721 and 671 rps (revolutions/s), respectively. The average rate of 60-nm beads was 381 rps, which is ∼13-fold faster than that expected from the steady-state ATPase turnover number. These results indicate that the F1 sector rotates much faster than expected from the bulk of ATPase activity, and that ∼10% of the F1 molecules are active on the millisecond time scale. Furthermore, the real ATP turnover number (number of ATP molecules converted to ADP and phosphate/s), as a single molecule, is variable during a short period. The ϵ subunit inhibited rotation and ATPase, whereas ϵ fused through its carboxyl terminus to cytochrome b562 showed no effect. The ϵ subunit significantly increased the pausing time during rotation. Stochastic fluctuation of catalysis may be a general property of an enzyme, although its understanding requires combining studies of steady-state kinetics and single molecule observation.

The Role of the ϵ Subunit in the Escherichia coli ATP Synthase THE C-TERMINAL DOMAIN IS REQUIRED FOR EFFICIENT ENERGY COUPLING

The role of the C-domain of the ϵ subunit of ATP synthase was investigated by fusing either the 20-kDa flavodoxin (Fd) or the 5-kDa chitin binding domain (CBD) to the N termini of both full-length ϵ and a truncation mutant ϵ88-stop. All mutant ϵ proteins were stable in cells and supported F1F0 assembly. Cells expressing the Fd-ϵ or Fd-ϵ88-stop mutants were unable to grow on acetate minimal medium, indicating their inability to carry out oxidative phosphorylation because of steric blockage of rotation. The other forms of ϵ supported growth on acetate. Membrane vesicles containing Fd-ϵ showed 23% of the wild type ATPase activity but no proton pumping, suggesting that the ATP synthase is intrinsically partially uncoupled. Vesicles containing CBD-ϵ were indistinguishable from the wild type in ATPase activity and proton pumping, indicating that the N-terminal fusions alone do not promote uncoupling. Fd-ϵ88-stop caused higher rates of uncoupled ATP hydrolysis than Fd-ϵ, and ϵ88-stop showed an increased rate of membrane-bound ATP hydrolysis but decreased proton pumping relative to the wild type. Both results demonstrate the role of the C-domain in coupling. Analysis of the wild type and ϵ88-stop mutant membrane ATPase activities at concentrations of ATP from 50 μm to 8 mm showed no significant dependence of the ratio of bound/released ATPase activity on ATP concentration. These results support the hypothesis that the main function of the C-domain in the Escherichia coli ϵ subunit is to reduce uncoupled ATPase activity, rather than to regulate coupled activity.

Genetic Fusions of Globular Proteins to the ε Subunit of theEscherichia coli ATP Synthase IMPLICATIONS FOR IN VIVO ROTATIONAL CATALYSIS AND ε SUBUNIT FUNCTION

The rotational mechanism of ATP synthase was investigated by fusing three proteins from Escherichia coli, the 12-kDa soluble cytochrome b 562, the 20-kDa flavodoxin, and the 28-kDa flavodoxin reductase, to the C terminus of the ε subunit of the enzyme. According to the concept of rotational catalysis, because ε is part of the rotor a large domain added at this site should sterically clash with the second stalk, blocking rotation and fully inhibiting the enzyme. E. colicells expressing the cytochrome b 562 fusion in place of wild-type ε grew using acetate as the energy source, indicating their capacity for oxidative phosphorylation. Cells expressing the larger flavodoxin or flavodoxin reductase fusions failed to grow on acetate. Immunoblot analysis showed that the fusion proteins were stable in the cells and that they had no effect on enzyme assembly. These results provide initial evidence supporting rotational catalysis in vivo. In membrane vesicles, the cytochromeb 562 fusion caused an increase in the apparent ATPase activity but a minor decrease in proton pumping. Vesicles bearing ATP synthase containing the larger fusion proteins showed reduced but significant levels of ATPase activity that was sensitive to inhibition by dicyclohexylcarbodiimide (DCCD) but no proton pumping. Thus, all fusions to ε generated an uncoupled component of ATPase activity. These results imply that a function of the C terminus of ε in F1F0 is to increase the efficiency of the enzyme by specifically preventing the uncoupled hydrolysis of ATP. Given the sensitivity to DCCD, this uncoupled ATP hydrolysis may arise from rotational steps of γε in the inappropriate direction after ATP is bound at the catalytic site. It is proposed that the C-terminal domain of ε functions to ensure that rotation occurs only in the direction of ATP synthesis when ADP is bound and only in the direction of hydrolysis when ATP is bound.

The b subunit of Escherichia coli ATP synthase.

The b subunit of ATP synthase is a major component of the second stalk connecting the F1and F0 sectors of the enzyme and is essential for normal assembly and function. The156-residue b subunit of the Escherichia coli ATP synthase has been investigated extensivelythrough mutagenesis, deletion analysis, and biophysical characterization. The two copies ofb exist as a highly extended, helical dimer extending from the membrane to near the top ofF1, where they interact with the δ subunit. The sequence has been divided into four domains:the N-terminal membrane-spanning domain, the tether domain, the dimerization domain, andthe C-terminal δ-binding domain. The dimerization domain, contained within residues 60–122,has many properties of a coiled-coil, while the δ-binding domain is more globular. Sites ofcrosslinking between b and the a, α, β, and δ subunits of ATP synthase have been identified,and the functional significance of these interactions is under investigation. The b dimer mayserve as an elastic element during rotational catalysis in the enzyme, but also directly influencesthe catalytic sites, suggesting a more active role in coupling.

Arrangement of Subunits in the Proteolipid Ring of the V-ATPase

The vacuolar ATPases (V-ATPases) are multisubunit complexes containing two domains. The V1 domain (subunits A–H) is peripheral and carries out ATP hydrolysis. The V0 domain (subunits a, c, c′, c″, d, and e) is membrane-integral and carries out proton transport. In yeast, there are three proteolipid subunits as follows: subunit c (Vma3p), subunit c′ (Vma11p), and subunit c″ (Vma16p). The proteolipid subunits form a six-membered ring containing single copies of subunits c′ and c″ and four copies of subunit c. To determine the possible arrangements of proteolipid subunits in V0 that give rise to a functional V-ATPase complex, a series of gene fusions was constructed to constrain the arrangement of pairs of subunits in the ring. Fusions containing c″ employed a truncated version of this protein lacking the first putative transmembrane helix (which we have shown previously to be functional), to ensure that the N and C termini of all subunits were located on the luminal side of the membrane. Fusion constructs were expressed in strains disrupted in c′, c″, or both but containing a wild copy of c to ensure the presence of the required number of copies of subunit c. The c-c″(ΔTM1), c″(ΔTM1)-c′, and c′-c constructs all complemented the vma– phenotype and gave rise to complexes possessing greater than 25% of wild-type levels of activity. By contrast, neither the c-c′, the c′-c″(ΔTM1), nor the c″(ΔTM1)-c constructs complemented the vma– phenotype. These results suggest that functionally assembled V-ATPase complexes contain the proteolipid subunits arranged in a unique order in the ring.

Mutations in the dimerization domain of the b subunit from the Escherichia coli ATP synthase: Deletions disrupt function but not enzyme assembly

The b subunit dimer of Escherichia coli ATP synthase serves essential roles as an assembly factor for the enzyme and as a stator during rotational catalysis. To investigate the functional importance of its coiled coil dimerization domain, a series of internal deletions including each individual residue between Lys-100 and Ala-105 (bΔK100-bΔA105), bΔK100-A103, and bΔK100-Q106 as well as a control bK100A missense mutation were prepared. All of the mutants supported assembly of ATP synthase, but all single-residue deletions failed to support growth on acetate, indicating a severe defect in oxidative phosphorylation, and bΔK100-Q106 displayed moderately reduced growth. The membrane-bound ATPase activities of these strains showed a related reduction in sensitivity to dicyclohexylcarbodiimide, indicative of uncoupling. Analysis of dimerization of the soluble constructs of bΔK100 and the multiple-residue deletions by sedimentation equilibrium revealed reduced dimerization compared with wild type for all deletions, with bΔK100-Q106 most severely affected. In cross-linking studies it was found that F1-ATPase can mediate the dimerization of some soluble b constructs but did not mediate dimerization of bΔK100 and bΔK100-Q106; these two forms also were defective in F1 binding analyses. We conclude that defective dimerization of soluble b constructs severely affects F1 binding in vitro, yet allows assembly of ATP synthase in vivo. The highly uncoupled nature of enzymes with single-residue deletions in b indicates that the b subunit serves an active function in energy coupling rather than just holding on to the F1 sector. This function is proposed to depend on proper, specific interactions between the b subunits and F1.

Tethering polypeptides through bifunctional PEG cross‐linking agents to probe protein function: Application to ATP synthase

Chemical crosslinking mediated by short bifunctional reagents has been widely used for determining physical relationships among polypeptides in multisubunit proteins, but less often for functional studies. Here we introduce the approach of tethering polypeptides by using bifunctional reagents containing a lengthy, flexible PEG linker as a form of crosslinking especially suited to functional analyses. The rotary molecular motor ATP synthase was used as a model subject. Single cysteine residues were introduced into selected positions of ATP synthase ϵ subunit, a component of the rotor subcomplex of the enzyme, and the unrelated maltose binding protein (MBP), then the two purified recombinant proteins were crosslinked by means of a dimaleimido‐PEG cross‐linking agent. Following purification, the ϵ‐PEG‐MBP was incorporated into membrane‐bound ATP synthase by reconstitution with ϵ‐depleted F1‐ATPase and membrane vesicles that had been stripped of endogenous F1. ATP synthase reconstituted using ϵ‐PEG‐MBP had reduced ATP hydrolytic activity that was uncoupled from the pumping of H+, indicating the physical blockage of rotation of the γϵc10 rotor by the conjugated MBP, whereas enzyme reconstituted with ϵ‐PEG was normal. These results directly demonstrate the feasibility of studying mechanistic features of molecular motors through PEG‐based conjugation of unrelated proteins. Since tethering polypeptides provides a means of maintaining proximity without directly specifying or modifying interactions, application of the general method to other types of protein functional studies is envisioned. Proteins 2008.