The role of residual phospholipids and copurified proteins in the reconstitution of bovine heart mitochondrial ATPase complex

The reactivation of mitochondrial ATPase by acidic and isoelectric phospholipids was studied comparatively with two purified enzyme preparations exhibiting different gel electrophoretic patterns: the preparation of Serrano et al. (1976, J. Biol. Chem. 251, 2453-2461) and the complex V of Galante et al. (1979, J. Biol. Chem. 254, 12372-12379). Isoelectric phosphatidylcholine liposomes showed marked differences in affinity for the two ATPase complexes and produced different maximal reactivations, whereas no significant differences were found with negatively charged liposomes. Analysis of residual phospholipids associated with the two ATPase preparations revealed a greater relative cardiolipin content in complex V. It is proposed that the different patterns of reactivation of the two ATPase preparations by isoelectric phospholipids result from different contents in residual cardiolipin and adenine nucleotide carrier.     

Localization of unassembled subunits of the mitochondrial ATPase in an assembly-defective yeast nuclear mutant

The yeast nuclear mutant, pet 936, has previously been shown to be defective in the assembly of a functional mitochondrial ATPase (Todd, R. D., McAda, P. C., and Douglas, M. G. (1979) J. Biol. Chem. 254, 11134-11141). In the present report, trypsin degradation and subunit-specific antibody binding have been used to localize subunits 1, 2, and 3 external to or associated with the outer aspect of the inner mitochondrial membrane in the mutant strain. A similar population of unassembled subunits was found in the parental strain as well. Isotope dilution experiments are compatible with those unassembled subunits being normal intermediates in the assembly pathway of the ATPase complex which are blocked from transport across the inner mitochondrial membrane in the mutant, pet 936.     

Assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar H(+)-ATPase.

Previous purification and characterization of the yeast vacuolar proton-translocating ATPase (H(+)-ATPase) have indicated that it is a multisubunit complex consisting of both integral and peripheral membrane subunits (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095; Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We have obtained monoclonal antibodies recognizing the 42- and 100-kDa polypeptides that were co-purified with vacuolar ATPase activity. Using these antibodies we provide further evidence that the 42-kDa polypeptide, a peripheral membrane protein, and the 100-kDa polypeptide, an integral membrane protein, are genuine subunits of the yeast vacuolar H(+)-ATPase. The synthesis, assembly, and targeting of three of the peripheral subunits (the 69-, 60-, and 42-kDa subunits) and two of the integral membrane subunits (the 100- and 17-kDa subunits) were examined in mutant yeast cells containing chromosomal deletions in the TFP1, VAT2, or VMA3 genes, which encode the 69-, 60-, and 17-kDa subunits, respectively. The steady-state levels of the various subunits in whole cell lysates and purified vacuolar membranes were assessed by Western blotting, and the intracellular localization of the 60- and 100-kDa subunits was also examined by immunofluorescence microscopy. The results suggest that the assembly and/or the vacuolar targeting of the peripheral subunits of the yeast vacuolar H(+)-ATPase depend on the presence of all three of the 69-, 60-, and 17-kDa subunits. The 100-kDa subunit can be transported to the vacuole independently of the peripheral membrane subunits as long as the 17-kDa subunit is present; but in the absence of the 17-kDa subunit, the 100-kDa subunit appears to be both unstable and incompetent for transport to the vacuole.     

Localization of subunit C (Vma5p) in the yeast vacuolar ATPase by immuno electron microscopy

Vacuolar ATPases (V1V0 -ATPases) function in proton translocation across lipid membranes of subcellular compartments. We have used antibody labeling and electron microscopy to define the position of subunit C in the vacuolar ATPase from yeast. The data show that subunit C is binding at the interface of the ATPase and proton channel, opposite from another stalk density previously identified as subunit H [Wilkens S., Inoue T., and Forgac M. (2004) Three-dimensional structure of the vacuolar ATPase - Localization of subunit H by difference imaging and chemical cross-linking. J. Biol. Chem. 279, 41942-41949]. A picture of the vacuolar ATPase stalk domain is emerging in which subunits C and H are positioned to play a role in reversible enzyme dissociation and activity silencing.     

Characterization of the functional coupling of bovine brain vacuolar-type H(+)-translocating ATPase. Effect of divalent cations,phospholipids, and subunit H (SFD)

Vacuolar-type H+-translocating ATPases (V-ATPases or V-pumps) are complex proteins containing multiple subunits and are organized into two functional domains: a peripheral catalytic sector V1 and a membranous proton channel V0. The functional coupling of ATP hydrolysis activity to proton transport in V-pumps requires a regulatory component known as subunit H (SFD) as has been shown both in vivo and in vitro (Ho, M. N., Hirata, R., Umemoto, N., Ohya, Y., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1993) J. Biol. Chem. 268, 18286-18292; Xie, X. S., Crider, B. P., Ma, Y. M., and Stone, D. K. (1994) J. Biol. Chem. 269, 25809-25815). Ca2+ is thought to uncouple V-pumps because it is found to support ATP hydrolysis but not proton transport, while Mg2+ supports both activities. The direct effect of phospholipids on the coupling of V-ATPases has not been reported, likely due to the fact that phospholipids are constituents of biological membranes. We now report that Ca2+-induced uncoupling of the bovine brain V-ATPase can be reversed by imposition of a favorable membrane potential. Furthermore we report a simple "membrane-free" assay system using the V0 proton channel-specific inhibitor bafilomycin as a probe to detect the coupling of V-ATPase under certain conditions. With this system, we have characterized the functional effect of subunit H, divalent cations, and phospholipids on bovine brain V-ATPase and have found that each of these three factors plays a critical role in the functional coupling of the V-pump.     

The phosphatidylglycerol/cardiolipin biosynthetic pathway is required for the activation of inositol phosphosphingolipid phospholipase C, Isc1p, during growth of Saccharomyces cerevisiae

Inositolsphingolipid phospholipase C (Isc1p) is the Saccharomyces cerevisiae member of the extended family of neutral sphingomyelinases that regulates the generation of bioactive ceramides. Recently, we reported that Isc1p is post-translationally activated in the post-diauxic phase of growth and that it localizes to mitochondria (Vaena de Avalos, S., Okamoto, Y., and Hannun, Y. A. (2004) J. Biol. Chem. 279, 11537-11545). In this study the in vivo mechanisms of activation and function of Isc1p were investigated. Deletion of ISC1 resulted in markedly lower growth in non-fermentable carbon sources. Interestingly, the growth defect of isc1Delta strains resembled that of pgs1Delta strains, lacking the committed step in the synthesis of phosphatidylglycerol (PG) and cardiolipin (CL), which were shown to activate Isc1p in vitro. Therefore, the role of Pgs1p in activation of Isc1p in vivo was investigated. The results showed that in the pgs1Delta strain, the growth-dependent activation of Isc1p was impaired as was the ISC1-dependent increase in the levels of phytoceramide during the post-diauxic phase, demonstrating that the activation of Isc1p in vivo is dependent on PGS1 and on the mitochondrial phospholipids PG/CL. Mechanistically, loss of Isc1p resulted in lower levels of mitochondrial cytochrome c oxidase subunits cox3p and cox4p, previously established targets of both PG and CL (Ostrander, D. B., Zhang, M., Mileykovskaya, E., Rho, M., and Dowhan, W. (2001) J. Biol. Chem. 276, 25262-25272), thus suggesting that Isc1p mediates at least some functions downstream of PG/CL. This study provides the first evidence for the mechanism of in vivo activation and function of Isc1p. A model with endogenous PG/CL as the in vivo activator of Isc1p is proposed.     

A gene encoding the proteolipid subunit of Sulfolobus acidocaldarius ATPase complex

An analysis of genes for the major two subunits of the membrane-associated ATPase from an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, suggested that it belongs to a different ATPase family from the F1-ATPase (Denda, K., Konishi, J., Oshima, T., Date, T., and Yoshida, M. (1988) J. Biol. Chem. 263, 17251-17254). In the same operon of the above two genes we found a gene encoding a very hydrophobic protein of 101 amino acids (Mr = 10,362). A proteolipid was purified from the membranes of this bacteria in which partial amino acid sequences matched with the sequence deduced from the gene. Significant amino acid sequence homology and a similar hydropathy profile appeared when the sequence was compared with the 8-kDa proteolipid subunit of F0F1-ATPases. It is about 30 amino acids larger than the 8-kDa proteolipid and has a small (11-amino acid) repeat sequence. However, it is distinct from the 16-kDa proteolipid subunit of an eukaryotic vacuolar H+-ATPase (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,5521-5524).     

Studies on the activation of rat liver pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase by adrenaline and glucagon. Role of increases in intramitochondrial Ca2+ concentration.

The association of different phospholipids with a lipid-depleted oligomycin-sensitive ATPase from bovine cardiac mitochondria [Serrano, Kanner & Racker (1976) J. Biol. Chem. 251, 2453-2461] has been examined using three approaches. First, reconstitution of the ATPase with different synthetic diacyl phospholipids resulted in a 2-10-fold stimulation of ATPase specific activity depending upon the particular phospholipid employed. The phospholipid headgroup region displayed the following order of ATPase reactivation potential: dioleoylphosphatidylglycerol greater than dioleoylphosphatidic acid greater than dioleoylphosphatidylcholine. Furthermore, the ATPase showed higher levels of specific activity when reconstituted with dioleoyl phospholipid derivatives compared with dimyristoyl derivatives. Second, examination of the phospholipid remaining associated with the lipid-depleted ATPase upon purification showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were present. No relative enrichment of any of these phospholipids (compared with their distribution in submitochondrial particles) was noted. Therefore, no preferential association between the ATPase and any one phospholipid could be found in the mitochondrial ATPase. Third, the sodium cholate-mediated phospholipid exchange procedure was employed for studying the phospholipid requirements of the ATPase. Replacement of about 50% of the mitochondrial phospholipid remaining with the lipid-depleted ATPase could be achieved utilizing either synthetic phosphatidic acid or phosphatidylcholine. Examination of the displaced mitochondrial phospholipid showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were replaced with equal facility.     

Proteolysis and orientation on reconstitution of the coated vesicle proton pump

We recently proposed a structural model for the ATP-dependent proton pump from clathrin-coated vesicles (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802). To test this model further, we have carried out additional structural analysis of the (H+)-ATPase in both the detergent-solubilized and reconstituted states in this and the following paper (Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 265, 967-973). The orientation of the reconstituted proton pump was determined by analyzing the effect of detergent on ATP hydrolysis and by quantitating the extent of labeling of luminally oriented subunits using a membrane-impermeant reagent. Greater than 90% of the reconstituted (H+)-ATPase is oriented with the cytoplasmic surface facing outward. Treatment of the reconstituted (H+)-ATPase with trypsin results in rapid cleavage of the 100-, 73-, 58-, 38-, and 34-kDa subunits and slower cleavage of the 40- and 33-kDa subunits, consistent with our previous results indicating that all of these polypeptides have some portion of their mass exposed to the cytoplasmic surface. The 19- and 17-kDa subunits, by contrast, appear resistant to cleavage by trypsin in both the detergent-solubilized and reconstituted states, consistent with their being buried extensively in the hydrophobic phase of the bilayer. Treatment of the enzyme with trypsin under conditions in which the 100-, 73-, 58-, 38-, and 34-kDa subunits have been cleaved results in a species which is virtually inactive with respect to proton transport but retains 50% of the original ATPase activity, suggesting that proteolysis has resulted in uncoupling of these two activities. Cleavage of both the 73- and 58-kDa subunits by trypsin at a site 1-2 kDa from the amino terminus is inhibited in the presence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP, consistent with the suggestion that both the 73- and 58-kDa subunits may be nucleotide binding proteins.     

Isolation of the structural genes for the alpha and beta subunits of the mitochondrial ATPase from the fission yeast Schizosaccharomyces pombe

The structural genes for the two major subunits of the mitochondrial ATPase were isolated among genomic clones from the yeast Schizosaccharomyces pombe by transformation and complementation of mutants unable to grow on glycerol and lacking either the alpha or the beta subunits. The plasmid pMa1 containing a 2.3-kilobase genomic insert transformed the mutant A23-13 lacking a detectable alpha subunit. The transformant grew on glycerol and contained an alpha subunit of normal electrophoretic mobility. The plasmid pMa2 containing a 5.4-kilobase genomic insert transformed the mutant B59-1 lacking the beta subunit. The transformant grew on glycerol and contained a beta subunit of normal mobility. The structural gene for the beta ATPase subunit for the fission yeast S. pombe was localized within the pMa2 insert by hybridization to a probe containing the beta ATPase gene from the budding yeast Saccharomyces cerevisiae (Saltzgaber, J., Kunapuli, S., and Douglas, M. G. (1983) J. Biol. Chem. 258, 11465-11470). The mRNAs which hybridized to pMa1 and pMa2 were translated by a reticulocyte lysate into polypeptides of Mr = 59,000 and 54,000, respectively. These genes products reacted with an anti-F1-ATPase serum and therefore correspond most probably to precursors of the alpha and beta subunits.     

Interaction between RNA polymerase and RapA, a bacterial homolog of the SWI/SNF protein family

Recently, we identified a novel Escherichia coli RNA polymerase (RNAP)-associated protein, an ATPase, called RapA (Sukhodolets, M. V. , and Jin, D. J. (1998) J. Biol. Chem. 273, 7018-7023). RapA is a bacterial homolog of SWI2/SNF2. We showed that RapA forms a stable complex with RNAP holoenzyme and that binding to RNAP holoenzyme stimulates the ATPase activity of RapA. We have further analyzed the interactions between purified RapA and the two forms of RNAP: core RNAP and RNAP holoenzyme. We found that RapA interacts with either form of RNAP. However, RapA exhibits higher affinity for core RNAP than for RNAP holoenzyme. Chemical cross-linking of the RNAP-RapA complex indicated that the RapA-binding sites are located at the interface between the alpha and beta' subunits of RNAP. Contrary to previously reported results (Muzzin, O., Campbell, E., A., Xia, L., Severinova, E., Darst, S. A., and Severinov, K. (1998) J. Biol. Chem. 273, 15157-15161), our in vivo analysis of a rapA null mutant suggested that RapA is not likely to be directly involved in DNA repair.     

Novel regulatory enhancer in the nuclear gene of the human mitochondrial ATP synthase beta-subunit

The system coordinating expressions of nuclear coded mitochondrial proteins was investigated by examination of the 5'-flanking region of the human mitochondrial ATP synthase beta-subunit gene. The promoter activity was measured by a transient expression of a chloramphenicol acetyltransferase (CAT) gene connected with various 5'-deletion mutants of the 5'-flanking region. In this experiment, at least two regions enhanced this promoter activity and at least one region repressed it. In one of the enhancing regions, a consensus sequence was found for the genes of other mitochondrial proteins such as those for cytochrome c1 (Suzuki, H., Hosokawa, Y., Nishikimi, M., and Ozawa, T. (1989) J. Biol. Chem. 264, 1368-1374) and the pyruvate dehydrogenase alpha-subunit (Maragos, C., Hutchison, W. M., Hayasaka, K., Brown, G. K., and Dahl, H.-H. M. (1989) J. Biol. Chem. 264, 12294-12298; Ohta, S., Endo, H., Matsuda, K., and Kagawa, Y. (1989) Ann. N. Y. Acad. Sci. 573, 458-460). The characteristics of this enhancing element were examined by introducing a synthetic oligonucleotide element into the CAT plasmid with a deleted enhancing element. The resulting plasmid showed full recovery of promoter activity, and this activity was independent of the orientation or location of the insert. Therefore, this is an enhancer that may be common to the nuclear genes of some mitochondrial proteins involved in energy transduction.     

Immunogold localization of the regulatory subunit of a type II cAMP- dependent protein kinase tightly associated with mammalian sperm flagella

We have shown previously that the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is an integral component of the mammalian sperm flagellum (Horowitz, J.A., H. Toeg, and G.A. Orr. 1984. J. Biol. Chem. 259:832-838; Horowitz, J.A., W. Wasco, M. Leiser, and G.A. Orr. 1988. J. Biol. Chem. 263:2098-2104). The subcellular localization of this flagellum-associated RII in bovine caudal epididymal sperm was analyzed at electron microscope resolution with gold-conjugated secondary antibody labeling techniques using anti-RII monoclonal antibodies. By immunoblot analysis, the flagellum-associated RII was shown to interact with mAb 622 which cross reacts with both neural and nonneural isoforms of RII. In contrast, a neural specific monoclonal antibody (mAb 526) failed to interact with flagellar RII. In the midpiece of the demembranated sperm tail, gold label after mAb 622 incubation was primarily associated with the outer mitochondrial membrane. Although almost all specific labeling in the midpiece can be assigned to the mitochondria, in the principal piece, there is some labeling of the fibrous sheath. Labeling of the outer dense fibers and the axoneme was sparse. Specific labeling was virtually absent in the sperm head. Sections of sperm tails incubated in the absence of primary antisera or with mAb 526 showed little labeling. A beta-tubulin monoclonal antibody localized only to the 9 + 2 axoneme. These results raise the possibility that a type II cAMP-dependent protein kinase located at the outer mitochondrial membrane plays a role in the direct cAMP stimulation of mitochondrial respiration during sperm activation.     

DNA polymerase III of Escherichia coli. Purification and identification of subunits.

DNA polymerase III, the core of the DNA polymerase III holoenzyme, has been purified 28,000-fold to 97% homogeneity from Escherichia coli HMS-83. The enzyme contains subunits: alpha, epsilon, and theta of 140,000, 25,000, and 10,000 daltons, respectively. The alpha subunit has been previously shown to be a component of both DNA polymerase III and the more complex DNA polymerase III holoenzyme (Livingston, D.M., Hinkle, D., and Richardson, C. (1975) J. Biol. Chem. 250, 461-469; McHenry, C., and Kornberg, A. (1977) J. Biol. Chem. 252, 6478-6484). It is demonstrated here that the epsilon and theta subunits are also subunits of the DNA polymerase III holoenzyme. Thus, the DNA polymerase III holoenzyme contains at least six different subunits. Our preparation has both the 3' leads to 5' and 5' leads to 3' exonuclease activities previously assigned to DNA polymerase III (Livingston, D., and Richardson, C. (1975) J. Biol. Chem. 250, 470-478).     

A 174-kilodalton ATPase/dATPase polypeptide and a glycoprotein of apparently identical molecular weight are common but distinct components of higher eukaryotic nuclear structural protein subfractions

A 174-kilodalton polypeptide of the Drosophila nuclear matrix-pore complex-lamina fraction has been identified as an ATPase/dATPase on the basis of direct UV photoaffinity labeling studies; a polypeptide with similar properties has been found in nuclear envelope fractions prepared from several vertebrate sources (Berrios, M., Blobel, G., and Fisher, P. A. (1983) J. Biol. Chem. 258, 4548-4555). This ATPase/dATPase polypeptide co-migrates on sodium dodecyl sulfate (SDS)-polyacrylamide gels with a glycoprotein also found in all of these fractions. In rat liver, this glycoprotein has been localized to the nuclear pore complex by means of immunoelectron microscopy (Gerace, L., Ottaviano, Y., and Kondor-Koch, C. (1982) J. Cell Biol. 95, 826-837). Following SDS denaturation and reduction/alkylation, chromatography of the Drosophila nuclear matrix-pore complex-lamina fraction on hydroxylapatite columns run in the presence of SDS results in the separation of two quantitatively major 174-kilodalton polypeptides. The peak of glycoprotein elution from the SDS-hydroxylapatite column correlates exactly with that of the early eluting 174-kilodalton species while the photolabeled ATPase/dATPase polypeptide co-chromatographs with the late eluting one. Identical results have been obtained with the rat liver nuclear envelope fraction. The chromatographically separated 174-kilodalton species from both organisms have been further distinguished through the use of polypeptide-specific antisera; finally, the glycoprotein purified from Drosophila embryos is fully sensitive to limited degradation by endoglycosidase H whereas the ATPase/dATPase polypeptide is completely resistant. We have thus established, using material obtained from two widely divergent higher eukaryotes, that the 174-kilodalton ATPase/dATPase is a quantitatively major nuclear matrix-pore complex-lamina component distinct from the nuclear pore complex glycoprotein of apparently identical molecular weight.     

A mammalian chromatin remodeling complex with similarities to the yeast INO80 complex

The mammalian Tip49a and Tip49b proteins belong to an evolutionarily conserved family of AAA+ ATPases. In Saccharomyces cerevisiae, orthologs of Tip49a and Tip49b, called Rvb1 and Rvb2, respectively, are subunits of two distinct ATP-dependent chromatin remodeling complexes, SWR1 and INO80. We recently demonstrated that the mammalian Tip49a and Tip49b proteins are integral subunits of a chromatin remodeling complex bearing striking similarities to the S. cerevisiae SWR1 complex (Cai, Y., Jin, J., Florens, L., Swanson, S. K., Kusch, T., Li, B., Workman, J. L., Washburn, M. P., Conaway, R. C., and Conaway, J. W. (2005) J. Biol. Chem. 280, 13665-13670). In this report, we identify a new mammalian Tip49a- and Tip49b-containing ATP-dependent chromatin remodeling complex, which includes orthologs of 8 of the 15 subunits of the S. cerevisiae INO80 chromatin remodeling complex as well as at least five additional subunits unique to the human INO80 (hINO80) complex. Finally, we demonstrate that, similar to the yeast INO80 complex, the hINO80 complex exhibits DNA- and nucleosome-activated ATPase activity and catalyzes ATP-dependent nucleosome sliding.     

A model for the structure of the yeast mitochondrial adenosine triphosphatase complex

Monodisperse, Triton-solubilized ATPase complex was treated with the reversible protein-protein cross-linker dithiobis(succinimidyl)propionate under conditions where only intracomplex cross-links were formed. The resulting products were analyzed by two-dimensional sodium dodecyl sulfate-acrylamide slab gel electrophoresis under reducing and oxidizing conditions. Using the observed major subunit-subunit cross-links and the previously published subunit stoichiometries (Todd, R., Griesenbeck, T., and Douglas, M. (1980) J. Biol. Chem. 255, 5461-5467), a model of the ATPase complex was constructed. The model is a closed structure which could be formed by self-limited assembly of the subunits. The model also has the novel features of a pseudo-mirror symmetry and clustering of mitochondrially and nuclearly coded subunits into two different domains.     

A membrane-bound protein inhibitor of the high affinity Ca ATPase in rat liver plasma membranes

The Ca ATPase from rat liver plasma membranes has been recently characterized and partially purified in our laboratory and was shown to depend on a membrane-bound protein activator (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215). In the present study, we report that a factor derived from ammonium sulfate washings of rat liver plasma membranes inhibits the partially purified enzyme activity measured in the presence of activator. This factor is a protein as judged by its sensitivity to heat and trypsin. A molecular weight of 29,000 was determined by sucrose gradient centrifugation and gel chromatography. The action of the inhibitor is due to a decrease in the maximal velocity of the enzyme reaction and is reversed by an excess of the activator associated with the enzyme. An important point in the mode of action of this inhibitor is its absolute dependence on magnesium, which most probably explains the difficulty in detecting the plasma membrane Ca ATPase when MgCl2 is added to the assay medium.     

Assembly of the F0 proton channel of the Escherichia coli F1F0 ATPase: low proton conductance of reconstituted Fo sectors synthesized and assembled in the absence of F1.

We have previously proposed that during assembly of the Escherichia coli F1F0 ATPase, the proton permeability of the Fo sector of the E. coli F1F0 ATPase is increased significantly by interactions with F1 subunits [Pati, S., & Brusilow, W.S.A. (1989) J. Biol. Chem 264, 2640-2644]. To test this model for Fo assembly, we purified F0 sectors synthesized in the presence and absence of F1 subunits and measured the abilities of these different preparations to bind purified F1 ATPase and to conduct protons when reconstituted into liposomes. The results of these studies demonstrated significant differences in proton-conducting abilities of the different Fo preparations. Fo sectors synthesized in the presence of F1 subunits were more permeable to protons than those synthesized in the absence of F1 subunits.