Active transport across the vacuolar components of the eukaryotic endomembrane system is energized by a specific vacuolar H+-ATPase. The amino acid sequences of the 70-and 60-kDa subunits of the vacuolar H+-ATPase are -25% identical to the .8 and a subunits, respectively, of the eubacterial-type FOFj-ATPases. We now report that the same vacuolar H+-ATPase subunits are -50% identical to the a and 13 subunits, respectively, of the sulfur-metabolizing Sulfolobus acidocaldarius, an archaebacterium (Archaeobacterium). Moreover, the homologue of an 88-amino acid stretch near the amino-terminal end of the 70-kDa subunit is absent from the FOFj-ATPase P subunit but is present in the a subunit of Sulfolobus. Since the two types of subunits (a and 13 subunits; 60-and 70-kDa subunits) are homologous to each other, they must have arisen by a gene duplication that occurred prior to the last common ancestor of the eubacteria, eukaryotes, and Sulfolobus. Thus, the phylogenetic tree of the subunits can be rooted at the site where the gene duplication occurred. The inferred evolutionary tree contains two main branches: a eubacterial branch and an eocyte branch that gave rise to Sulfolobus and the eukaryotic host cell. The implication is that the vacuolar H+-ATPase of eukaryotes arose by the internalization of the plasma membrane H+-ATPase of an archaebacterial-like ancestral cell.Recently, attention has focused on the evolutionary relationships among the H+-ATPases, particularly the F0F1-ATPases (F-type) and vacuolar (V-type) H+-ATPases. F-and VATPases exhibit a number of structural and functional similarities (1-4). Both are large, multisubunit enzymes (=500 kDa) composed of a water-soluble catalytic sector and an integral membrane proton channel complex. Each hydrophilic sector contains three copies of the catalytic subunit (F-ATPase (3 subunit or V-ATPase 70-kDa subunit), three copies of a regulatory subunit (F-ATPase a subunit or V-ATPase 60-kDa subunit), and one copy each of several minor subunits (4). Sequences obtained for several eukaryotic V-ATPase 70-and 60-kDa subunits confirmed that the Fand V-type H+-ATPases are indeed homologous (5-9). However, the low overall similarity (25%) and the presence of a large stretch of nonhomologous sequence in the 70-kDa subunit (5) suggest that they diverged early in evolution. Consistent with this view, sequences obtained for the two major subunits of the membrane H+-ATPase of Sulfolobus acidocaldarius, an archaebacterium (Archaeobacterium), indicated that the "archaebacterial-type" H+-ATPase is only distantly related to the eubacterial-type F-ATPases (10, 11). In this joint communication from four of the laboratories involved, we show that the H+-ATPase of S. acidocaldarius belongs in the V-ATPase class of proton pumps. The implications for the origin of eukaryotes are discussed. MATERIALS AND METHODSTo determine the evolutionary relationships among the different H+-ATPases, protein or DNA sequences coding for the two major subunits or parts of these subunits were aligned, ...

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We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca 2؉ -ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca 2؉ -ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca 2؉ /H ؉ exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 m from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.

The vacuolar H؉ -ATPase is inhibited with high specificity and potency by bafilomycin and concanamycin, macrolide antibiotics with similar structures. We previously reported that mutation at three residues in subunit c of the vacuolar ATPase from Neurospora crassa conferred strong resistance to bafilomycin but little or no resistance to concanamycin (Bowman, B. J., and Bowman, E. J. (2002) J. Biol. Chem. 277, 3965-3972). We have identified additional mutated sites in subunit c that confer resistance to bafilomycin. Furthermore, by subjecting a resistant mutant to a second round of mutation we isolated strains with increased resistance to both bafilomycin and concanamycin. In all of these strains the second mutation is also in subunit c, suggesting it forms at least part of the concanamycin binding site. Sitedirected mutagenesis of the gene encoding subunit c in Saccharomyces cerevisiae showed that single mutations in each of the residues identified in one of the double mutants of N. crassa conferred resistance to both bafilomycin and concanamycin. Mutations at the corresponding sites in the VMA11 and VMA16 genes of S. cerevisiae, which encode the c and c؆ subunits, did not confer resistance to the drugs. In all, nine residues of subunit c have been implicated in drug binding. The positions of these residues support a model in which the drug binding site is a pocket formed by helices 1, 2, and 4. We hypothesize that the drugs inhibit by preventing the rotation of the c subunits. Vacuolar proton-translocating ATPases (V-ATPases)1 are large, complex enzymes responsible for acidification of many internal compartments in eukaryotic cells. They also occur on plasma membranes of specialized cells, where they acidify the surrounding milieu. Numerous physiological processes depend on the activity of V-ATPases, including protein processing, transport of metabolites, receptor-mediated endocytosis, neurotransmitter uptake, and apoptosis (1, 2). V-ATPases are implicated as a contributing factor in multiple diseases such as osteoporosis (3), deafness (4), and cancer (5). Because of its vital role in so many processes and diseases, the V-ATPase is an attractive target for the development of therapeutic agents (6 -10).The bafilomycins and concanamycins have been particularly important in elucidating the physiological role of V-ATPases and are under investigation as possible lead compounds for drug therapy directed toward this enzyme (9, 11). These macrolide antibiotics, which are very similar in structure, are specific, highly potent inhibitors of all eukaryotic V-ATPases in vitro and in vivo (12)(13)(14). Despite the widespread use of these compounds, however, our understanding of how they inhibit is rudimentary. Early reports indicated that bafilomycin and concanamycin acted on the Vo segment of the enzyme (15-18). Using a radiolabeled derivative of concanamycin, Huss et al. (19) identified subunit c as the site bound by the inhibitor in the Manduca sexta enzyme.We have used a genetic approach to identify specific amino aci...

Bafilomycin A1, a potent inhibitor of vacuolar H؉ -ATPases (V-ATPase), inhibited growth of Neurospora crassa in medium adjusted to alkaline pH. Ninety-eight mutant strains were selected for growth on medium (pH 7.2) containing 0.3 or 1.0 M bafilomycin. Three criteria suggested that 11 mutant strains were altered in the V-ATPase: 1) these strains accumulated high amounts of arginine when grown at pH 5.8 in the presence of bafilomycin, 2) the mutation mapped to the locus of vma-3, which encodes the proteolipid subunit c of the V-ATPase, and 3) V-ATPase activity in purified vacuolar membranes was resistant to bafilomycin. Sequencing of the genomic DNA encoding vma-3 identified the following mutations: T32I (two strains), F136L (two strains), Y143H (two strains), and Y143N (five strains). Characterization of V-ATPase activity in the four kinds of mutant strains showed that the enzyme was resistant to bafilomycin in vitro, with half-maximal inhibition obtained at 80 -400 nM compared with 6.3 nM for the wildtype enzyme. Surprisingly, the mutant enzymes showed only weak resistance to concanamycin. Interestingly, the positions of two mutations corresponded to positions of oligomycin-resistant mutations in the c subunit of F 1 F 0 -ATP synthases (F-ATPases), suggesting that bafilomycin and oligomycin utilize a similar binding site and mechanism of inhibition in the related F-and V-ATPases.The vacuolar (H ϩ )-ATPase (V-ATPase) 1 is a large, complex enzyme that couples the hydrolysis of ATP to the transport of protons across membranes. In eucaryotic cells, this enzyme plays a role in many physiological processes. It is present in several types of cellular organelles such as vacuoles, lysosomes, coated vesicles, Golgi, and secretory vesicles (reviewed in Refs. 1-3), and it is also the major proton pump in the plasma membrane of specialized acid-secreting cells such as osteoclasts and kidney intercalated cells and the intestinal epithelia cells of some insects (4). The role of the V-ATPase in physiological processes has often been examined by measuring the effects of bafilomycin and concanamycin, macrolide antibiotics that are potent inhibitors of the enzyme (5). Effective at nanomolar concentrations in vitro, these two drugs also inhibit the VATPase in living cells, although higher concentrations, typically 0.1-10.0 M, are required (6). At low concentrations, the two antibiotics appear to be highly specific for V-ATPases; at 10,000-fold higher concentrations, they inhibit some P-type ATPases in vitro (7,8).Because of their effectiveness and specificity in vivo, bafilomycin and concanamycin are attractive candidates for development as therapeutic agents (9, 10). For example, considerable effort has been made to develop bafilomycin derivatives for the treatment of osteoporosis (11-14). Bafilomycin and concanamycin are also potent anti-tumor agents that exhibit significant cell line specificity (15). Thus, derivatives of these inhibitors might be effective in treating cancer. The pharmacological potential of these drugs has pr...

The total synthesis and stereochemical assignment of the potent antitumor macrolide lobatamide C, as well as synthesis of simplified lobatamide analogues, is reported. Cu(I)-mediated enamide formation methodology has been developed to prepare the highly unsaturated enamide side chain of the natural product and analogues. A key fragment coupling employs base-mediated esterification of a beta-hydroxy acid and a salicylate cyanomethyl ester. Three additional stereoisomers of lobatamide C have been prepared using related synthetic routes. The stereochemistry at C8, C11, and C15 of lobatamide C was assigned by comparison of stereoisomers and X-ray analysis of a crystalline derivative. Synthetic lobatamide C, stereoisomers, and simplified analogues have been evaluated for inhibition of bovine chromaffin granule membrane V-ATPase. The salicylate phenol, enamide NH, and ortho-substitution of the salicylate ester have been shown to be important for V-ATPase inhibitory activity.

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