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.