Various membrane ATPases have been tested for their sensitivity to baflomycin Al, a macrolide antibiotic. F1Fo ATPases from bacteria and mitochondria are not affected by this antibiotic. In contrast, ElE2 ATPases-e.g., the K+-dependent (Kdp) ATPase from Escherichia coli, the Na4+,K+ -ATPase from ox brain, and the Ca2+-ATPase from sarcoplasmic reticulum-are moderately sensitive to this inhibitor. Finally, membrane ATPases from Neurospora vacuoles, chromaffin granules, and plant vacuoles are extremely sensitive. and, in the case of the Na4 ,K4-ATPase, by ouabain (5-11).The vacuolar ATPases appear to hydrolyze ATP, generating a proton gradient that is used for acidification of compartments within cells (12,17,22). This group of ATPases has been distinguished from the other two by virtue of its inhibitor specificity. The vacuolar ATPases are not inhibited by azide, oligomycin, vanadate, or ouabain. Instead, inhibitors that have proved useful for these enzymes include (i) N,N'-dicyclohexylcarbodiimide, which also inhibits F1F0 and E1E2 ATPases, (ii) N-ethylmaleimide, which inhibits E1E2 enzymes (23) and at least one F1F0 ATPase (24), and (iii) NO3 -, which is effective only at millimolar concentrations or greater. Thus, no potent specific inhibitor of vacuolar ATPases has yet been identified.The bafilomycins A1, B1, C1, and D, macrolide antibiotics with a 16-membered lactone ring, were isolated from Streptomyces sp. (25). These compounds inhibited growth of Gram-positive bacteria and fungi in a disc diffusion assay. In addition, it has been reported that bafilomycin C1 inhibits the enzymatic activity of the Na4, 27). In this communication, we compare the effects of bafilomycin A1 on representative enzymes of the three classes of ATPases. The results show that bafilomycin A1 is useful for distinguishing among the different types of ATPases and that it is an extremely potent inhibitor of the vacuolar ATPases.

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, ...

Various ATPases have been tested for their sensitivity to naturally occurring unusual macrolides and their chemically modified derivatives, which are structurally related to bafilomycin A1 (1), the first specific inhibitor of vacuolar ATPases. The structure-activity study showed that in general the concanamycins, 18-membered macrolides, are better and more specific inhibitors than the bafilomycins of this class of membrane-bound ATPases. The additional carbohydrate residue is not responsible for the improved activity. The importance of an intact hemiketal ring, which is part of an intramolecular hydrogen-bonding network, and the effects of the size of the macrolactone ring are discussed. The structurally related elaiophylin (13), a C2-symmetric macrodiolide antibiotic, proved to be inactive on vacuolar ATPases but still retained its inhibitory effect on P-type ATPases.

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