The proton-translocating ATPase of the yeast vacuole is an enzyme complex consisting of a large peripheral membrane sector (V 1 ) and an integral membrane sector (V 0 ), each composed of multiple subunits. The V 1 sector contains subunits that hydrolyze ATP, whereas the V 0 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 V 1 but also implicated in interactions with the V 0 . 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 V 0 sector, our results indicate that these subunits behave primarily as canonical V 1 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 V 1 during later stages of V 1 assembly. This is the first demonstration of interdependence between ATPase subunits for structural stability.The vacuolar proton-translocating ATPases are a family of organellar proton pumps found throughout the endomembrane system of eukaryotic cells. The V-ATPases 1 are related to the F-type ATP synthases of mitochondria, chloroplasts, and bacterial membranes. The enzymes of both families have similar subunit stoichiometries (1), similar structures based on electron micrographic imaging (2, 3), and similar mechanisms (4). Furthermore, the major catalytic subunits (A and B in the V-type; ␤ and ␣ in the F-type) show primary sequence homology of about 30%, whereas the principal proton-translocating subunits, the c subunits or proteolipids, also show high sequence homology (5-7). The intermediate peripheral sector subunits and the remaining integral sector subunits show little or no homology between the two families. This suggests an overall conservation of gross structure with a low tolerance for deviation at the sites mediating the two principal activities: ATP hydrolysis (or synthesis) and proton translocation. In contrast, the intermediate subunits that couple these activities have greater evolutionary flexibility.The subunits coupling the F 1 peripheral sector to the F O membrane sector, ␥, ␦, ⑀, and the hydrophilic portion of F O -b, are described as "core" or "stalk" subunits because they reside in the midst of the catalytic subunits and appear to bridge physically the two sectors as seen in electron micrographic images. The subunits presumably bridging the V 1 and V 0 sectors, C (Vma5p), D (Vma8p), E (Vma4p), F (Vma7p), G (Vma10p), H (Vma13p), d (Vma6p), and perhaps hydrophilic portions of Vph1p, can therefore also be thought of as stalk or core subunits of the V-ATPase.We have id...

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 (12C) or stable-isotope-labeled (13C) 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 13C-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.

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, ⌬vma3 and ⌬vma5 strains, we see partial complexes in the ⌬vma4 and ⌬vma7 strains. All larger complexes are lost in the ⌬vma1, ⌬vma2, and ⌬vma8 strains. We designate the large complex seen in wild-type cells containing at least subunits Vma1p, Vma2p, Vma4p, Vma7p, and Vma8p as the definitive V 1 complex.The V-type proton-translocating ATPase is the cornerstone of the yeast vacuole. This multisubunit membrane-bound enzyme converts the energy of ATP into a proton electrochemical gradient that is essential for the majority of vacuolar functions, including ion homeostasis, accumulation of amino acids, and the correct targeting of vacuolar resident proteins (1-3). Enzymes of the V-ATPase class are found throughout the biological world serving many functions, oftentimes at different subcellular locations or with tissue-dependent activities (4).Many of the genes encoding ATPase subunits, as well as genes necessary for vacuolar acidification, have been identified (4, 5). Null mutations in the VMA (vacuolar membrane ATPase) genes result in a conditional phenotype characterized by several different traits. Growth of vma mutants is severely inhibited in media buffered to pH 7.0 or higher; best growth is obtained in media buffered to pH 5.5 (6). These mutants are sensitive to high concentrations of Ca 2ϩ (Ն50 mM) in the media (7). In vma strains that are also ade2, the reddish color caused by accumulation of fluorescent amino-imidazole ribotide conjugates in the vacuole is diminished, providing a convenient visual screen for ATPase mutants (8). Deacidification of the vacuolar lumen, which is normally maintained at pH 6 in wild-type cells, can be determined by direct fluorescent ratio measurements (9) and has been used as a screen for vacuolar ph mutants (5). Screens utilizing these characteristics have revealed the genes catalogued in Table I. Each of these genes is essential for vacuolar ATPase activity and most are required for proper assembly of the complete V-ATPase complex.Biochemical analyses have begun to elucidate the assembly and regulation of the enzyme (10, 11). The vacuolar ATPase is divided into two subcomplexes: a membrane-bound V 0 complex, which is responsible for the translocation of protons, and a peripheral membrane V 1 complex, which contains the ATPhydrolyzing subunits. In yeast, more extensive biochemical studies have lagged behind the more readily achieved genetic analyses. It has b...

Oligomeric assembly is a fundamental aspect of many complex enzymes. Using our native gel technique for examining subcomplexes of the V-ATPase V 1 sector, we have developed an in vitro reconstitution assay for assembly of this complex. Assembly of complex II, the soluble V 1 complex observed in native gels, is dependent upon the presence of divalent cations and physiological temperatures. Assembly of soluble V 1 can occur in a stepwise fashion from smaller subcomplexes found in some strains deleted for V-ATPase subunits. Specifically, V 1 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.Many enzymes are oligomeric and require the coordinated assembly of numerous individual subunits. The vacuolar proton-translocating ATPase (V-ATPase) 1 is such a multisubunit enzyme, comprised of over a dozen different proteins both in and on the membranes of vacuoles, lysosomes, tonoplasts, and related compartments (1). This enzyme is also found at plasma membranes in certain species and tissues. More complex than its evolutionary cousin, the F-type ATP synthase, the V-ATPase is composed of over 22 individual protein subunits that are the product of at least 12 unique genes, some of which have multiple isoforms in higher eukaryotes. Because interactions between these subunits are fundamental to both the assembly and the energy-transducing mechanism of this enzyme, we imagine that the pathway of assembly might be related to the mechanism of the enzyme.We have utilized native PAGE to reveal a myriad of partial V-ATPase complexes (Table I) found at steady state in the cytoplasm of both wild-type yeast as well as in deletion mutants of specific ATPase subunits encoded by the VMA and VPH gene families (2).2 In wild-type yeast, as well as strains lacking one or more integral membrane sector subunits (e.g. vma3⌬ and vph1⌬stv1⌬), we observe a large stable complex (II; 576 Ϯ 96 kDa) that we believe is the soluble cytoplasmic form of V 1 . This complex contains at least Vma1p (A subunit), Vma2p (B subunit), Vma4p (E subunit), Vma7p (F subunit), Vma8p (D subunit), and Vma10p (G subunit), with a probable stoichiometry of A 3 B 3 DEFG. This complex is also present in vma5⌬ (Vma5p is the peripheral subunit C), indicating Vma5p is not necessary for assembly of the V 1 . In all strains except vma4⌬ and vma10⌬, we observe a small complex (III; 96 Ϯ 28 kDa) consisting of, minimally, Vma4p and Vma10p. In vma4⌬ and vma10⌬, we observe an intermediate complex (IV; 317 Ϯ 49 kDa) that contains all of the above proteins except for Vma4p and Vma10p, ...

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