Stoichiometry of energy coupling by proton-translocating ATPases: a history of variability.

One of the central energy-coupling reactions in living systems is the intraconversion of ATP with a transmembrane proton gradient, carried out by proton-translocating F- and V-type ATPases/synthases. These reversible enzymes can hydrolyze ATP and pump protons, or can use the energy of a transmembrane proton gradient to synthesize ATP from ADP and inorganic phosphate. The stoichiometry of these processes (H(+)/ATP, or coupling ratio) has been studied in many systems for many years, with no universally agreed upon solution. Recent discoveries concerning the structure of the ATPases, their assembly and the stoichiometry of their numerous subunits, particularly the proton-carrying proteolipid (subunit c) of the F(O) and V(0) sectors, have shed new light on this question and raise the possibility of variable coupling ratios modulated by variable proteolipid stoichiometries.

Analysis of estrogen receptor interaction with tertiary-structured estrogen responsive elements.

An initial crucial step in estrogen activation of gene expression is the interaction of the estrogen receptor with a specific nucleotide sequence [estrogen responsive element (ERE)]. Previously, we found that the estrogen receptor binds preferentially and with high affinity to the lower strand of the rat prolactin imperfect ERE which contains tertiary structure (Lannigan DA and Notides AC, Proc Natl Acad Sci USA 86: 863-867, 1989). Using perfect and imperfect EREs from the upstream region of the chicken vitellogenin II gene, we have now extended our findings and have determined that the estrogen receptor preferentially interacts with either perfect or imperfect EREs which contain tertiary structure. A similar structure is present in a synthetic 42 bp oligonucleotide corresponding to the lower strand of a perfect ERE with flanking sequences from the rat prolactin ERE. Moreover, deviations from the ERE consensus sequence decrease the binding of the estrogen receptor to the tertiary-structured ERE. We also have determined that ERE flanking sequences contribute to the affinity of the receptor for the tertiary-structured ERE. Furthermore, ERE flanking sequences can influence the types of interactions that the estrogen receptor makes with the tertiary-structured ERE.

Characterization of reconstituted Fo from wild-type Escherichia coli and identification of two other fluxes co-purifying with Fo.

We purified the ATPase Fo sector from a nonoverexpressing strain of Escherichia coli, reconstituted it into lipid vesicles made of either asolectin or two different mixtures of purified lipids, and measured proton flux through the reconstituted proton channel. We measured single-channel conductances and found that Fo activity depends on both lipids and reconstitution methods. In asolectin vesicles, Fo has a single-channel conductance of about 0.2 fS. Additionally, the relatively impure Fo prepared from cells carrying single-copy ATPase genes allowed us to observe two other fluxes, a nonselective cation leak (C(L)) and a slow H+ flux (Hs). Unlike the Fo flux, these fluxes could not be blocked by the Fo inhibitor DCCD. The C, reduces the total apparent trapped volume inside vesicles and therefore must equilibrate both H+ and K+ in the vesicles that contain it. When reconstituted into bilayers, these Fo preparations displayed a 120 pS cation channel with characteristics consistent with C(L) flux. The Hs conducts only H+ but at a slower rate than the Fo. We were therefore able to: 1) quantitate the single-channel conductance of the Fo, 2) demonstrate that our Fo purification method co-purified other membrane proteins that have ion-conduction properties, and 3) show that certain lipids are necessary for functional reconstitution of Fo.

Multisubstrate isotope labeling and metagenomic analysis of active soil bacterial communities.

Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon ((12)C) or stable-isotope-labeled ((13)C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the (13)C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. Importance: The ability to identify genes based on function, instead of sequence homology, allows the discovery of genes that would not be identified through sequence alone. This is arguably the most powerful application of metagenomics for the recovery of novel genes and a natural partner of the stable-isotope-probing approach for targeting active-yet-uncultured microorganisms. We expanded on previous efforts to combine stable-isotope probing and metagenomics, enriching microorganisms from multiple soils that were active in degrading plant-derived carbohydrates, followed by construction of a cellulose-based metagenomic library and recovery of glycoside hydrolases through functional metagenomics. The major advance of our study was the discovery of active-yet-uncultivated soil microorganisms and enrichment of their glycoside hydrolases. We recovered positive cosmid clones in a higher frequency than would be expected with direct metagenomic analysis of soil DNA. This study has generated an invaluable metagenomic resource that future research will exploit for genetic and enzymatic potential.

Reconstitution in vitro of the V1 complex from the yeast vacuolar proton-translocating ATPase. Assembly recapitulates mechanism.

Oligomeric assembly is a fundamental aspect of many complex enzymes. Using our native gel technique for examining subcomplexes of the V-ATPase V1 sector, we have developed an in vitro reconstitution assay for assembly of this complex. Assembly of complex II, the soluble V1 complex observed in native gels, is dependent upon the presence of divalent cations and physiological temperatures. Assembly of soluble V1 can occur in a stepwise fashion from smaller subcomplexes found in some strains deleted for V-ATPase subunits. Specifically, V1 can be assembled directly from complex III (subunits E and G) with complex IV (subunits A, B, D, and F) without prior disassembly of complex IV. The formation of complex III in vivo is also shown to be essential and could not be achieved in vitro. Assembly from simpler precursors is possible and is enhanced by added ATP. Assembly can be blocked by N-ethylmaleimide in a Vma1p (subunit A)-specific manner. From these data, we extend our previous model to consider an assembly pathway whose steps reflect the catalytic mechanism of the Boyer binding-change model.

Purification and ligand binding of EmrR, a regulator of a multidrug transporter.

EmrR, the repressor of the emrRAB operon of Escherichia coli, was purified to 95% homogeneity. EmrR was found to bind putative ligands of the EmrAB pump-2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and carbonyl cyanide p-(trifluoro-methoxy)phenylhydrazone-with affinities in the micromolar range. Equilibrium dialysis experiments suggested one bound ligand per monomer of the dimeric EmrR.

A functional His-tagged c subunit of the Escherichia coli F-type ATPase/Synthase.

The c subunit of the Escherichia coli F0 has been tagged with a hexahistidine motif at its C-terminus. The tagged subunit is capable of forming functional F0 complexes that translocate protons in the absence of the F1 complex. In the presence of F1, the two sectors associate and display all biochemical activities of the wildtype enzyme: DCCD-inhibitable ATPase activity, ATP synthase activity, and ATP-dependent proton pumping. The enzyme can be solubilized and purified as an intact complex under native conditions on immobilized-metal affinity chromatography (IMAC) resin. The purified complex can be reincorporated into liposomes and demonstrates ATP-dependent proton pumping activity. Hexahistine tags placed at the N-terminus, in contrast, were all inactive. These experiments demonstrate the feasibility of tagging the c subunit for further studies of the F0 and suggest an important role for the N-terminus of the c subunit in either assembly or function of the protein.

Resolution of subunit interactions and cytoplasmic subcomplexes of the yeast vacuolar proton-translocating ATPase.

The vacuolar proton-translocating ATPase is the principal energization mechanism that enables the yeast vacuole to perform most of its physiological functions. We have undertaken an examination of subunit-subunit interactions and assembly states of this enzyme. Yeast two-hybrid data indicate that Vma1p and Vma2p interact with each other and that Vma4p interacts with itself. Three-hybrid data indicate that the Vma4p self-interaction is stabilized by both Vma1p and Vma2p. Native gel electrophoresis reveals numerous partial complexes not previously described. In addition to a large stable cytoplasmic complex seen in wild-type, Deltavma3 and Deltavma5 strains, we see partial complexes in the Deltavma4 and Deltavma7 strains. All larger complexes are lost in the Deltavma1, Deltavma2, and Deltavma8 strains. We designate the large complex seen in wild-type cells containing at least subunits Vma1p, Vma2p, Vma4p, Vma7p, and Vma8p as the definitive V1 complex.

V1-situated stalk subunits of the yeast vacuolar proton-translocating ATPase.

The proton-translocating ATPase of the yeast vacuole is an enzyme complex consisting of a large peripheral membrane sector (V1) and an integral membrane sector (V0), each composed of multiple subunits. The V1 sector contains subunits that hydrolyze ATP, whereas the V0 sector contains subunits that translocate protons across the membrane. Additional subunits in both sectors couple these activities. Here we have continued our examination of intermediate subunits primarily associated with the V1 but also implicated in interactions with the V0. Interactions between Vma7p (F) and Vma8p (D) and between Vma4p (E) and Vma10p (G) are described. Although Vma7p and Vma10p have been observed to interact with the V0 sector, our results indicate that these subunits behave primarily as canonical V1 sector subunits. We categorize these four subunits as "stalk" subunits to distinguish them from the known catalytic (A and B) and proton-translocating (c, c', and Vma16p) subunits and to highlight their intermediate nature. Furthermore, we show that the in vivo stability of Vma4p is dependent upon interaction with Vma10p. This may be important in the regulation of assembly, since these two subunits add to the V1 during later stages of V1 assembly. This is the first demonstration of interdependence between ATPase subunits for structural stability.