Functional reassembly of the coated vesicle proton pump

We have shown previously that treatment of the coated vesicle proton-translocating adenosine triphosphatase (H(+)-ATPase) with chaotropic agents results in the release of a set of peripheral polypeptides which includes the 73-, 58-, 40-, 34-, and 33-kDa subunits (Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 265, 967-973), with a coordinate loss of H(+)-ATPase activity. In the present paper we report the functional reassembly of the coated vesicle proton pump following dissociation of the peripheral subunits. Reassembly was demonstrated by restoration of ATP-driven proton transport using both native membranes and reconstituted vesicles and by Western blot analysis using a monoclonal antibody specific for the 73-kDa subunit. Reassembly occurs by attachment of a peripheral subcomplex containing the 73-, 58-, 34-, and 33-kDa subunits together with the 40-kDa polypeptide. The reassembled H(+)-ATPase, like the native proton pump, is inhibited by N-ethylmaleimide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and N,N'-dicyclohexylcarbodiimide. Reassociation shows a biphasic time dependence, with restoration of 50-60% of the starting proton transport activity in the 1st h followed by recovery of a further 20-30% of the activity after 24 h. Reassembly also shows a marked dependence on protein concentration but, unlike solubilization of the intact H(+)-ATPase complex, does not require the presence of glycerol. Despite the ability of nucleotides to promote dissociation of the peripheral complex by chaotropic agents, reassociation is not blocked by the presence of 1 mM ATP. These results thus provide the first evidence for functional reassembly of a vacuolar H(+)-ATPase complex and should be useful in further analysis of the role of individual subunits in the assembly and activity of these ATP-driven proton pumps.     

Dissociation, cross-linking, and glycosylation of the coated vesicle proton pump

In order to refine further our structural model of the coated vesicle (H+)-ATPase (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802), we have extended our structural analysis to identify peripheral and glycosylated subunits of the pump as well as to identify subunits which are in close proximity in the native (H+)-ATPase complex. Treatment of the purified, reconstituted (H+)-ATPase with 0.30 M KI in the presence or absence of ATP or MgATP results in the release of the 73-, 58-, 40-, 34-, and 33-kDa subunits, leaving behind the 100-, 38-, 19-, and 17-kDa subunits in the membrane. Because the former group of polypeptides is released from the membrane in the absence of detergent, they correspond to peripheral membrane proteins. To determine which subunits are in close proximity, cross-linking of the purified (H+)-ATPase was carried out using the cleavable, bifunctional amino reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) followed by two-dimensional gel electrophoresis. These studies indicate that contact regions exist between the 73- and 58-kDa subunits as well as between the 17-kDa subunit and the 40-, 34-, and 33-kDa subunits. To test for glycosylation of the (H+)-ATPase, the detergent-solubilized complex was treated with neuraminidase followed by electrophoresis and blotting using a peanut lectin/horseradish peroxidase conjugate. Galactose-inhibitable staining of the 100-kDa subunit, together with affinity chromatography of the intact (H+)-ATPase on peanut lectin agarose, indicates that the 100-kDa subunit is glycosylated, most likely at a site exposed on the luminal side of the membrane. These results, together with those presented in the preceding paper (Adachi, I., Arai, H., Pimental, R., and Forgac, M. (1990) J. Biol. Chem. 265, 960-966), were used in the construction of a refined model of the coated vesicle (H+)-ATPase.     

Reconstitution in vitro of V1 complex of Thermus thermophilus V-ATPase revealed that ATP binding to the A subunit is crucial for V1 formation

Vacuolar-type H(+)-ATPase (V-ATPase or V-type ATPase) is a multisubunit complex comprised of a water-soluble V(1) complex, responsible for ATP hydrolysis, and a membrane-embedded V(o) complex, responsible for proton translocation. The V(1) complex of Thermus thermophilus V-ATPase has the subunit composition of A(3)B(3)DF, in which the A and B subunits form a hexameric ring structure. A central stalk composed of the D and F subunits penetrates the ring. In this study, we investigated the pathway for assembly of the V(1) complex by reconstituting the V(1) complex from the monomeric A and B subunits and DF subcomplex in vitro. Assembly of these components into the V(1) complex required binding of ATP to the A subunit, although hydrolysis of ATP is not necessary. In the absence of the DF subcomplex, the A and B monomers assembled into A(1)B(1) and A(3)B(3) subcomplexes in an ATP binding-dependent manner, suggesting that ATP binding-dependent interaction between the A and B subunits is a crucial step of assembly into V(1) complex. Kinetic analysis of assembly of the A and B monomers into the A(1)B(1) heterodimer using fluorescence resonance energy transfer indicated that the A subunit binds ATP prior to binding the B subunit. Kinetics of binding of a fluorescent ADP analog, N-methylanthraniloyl ADP (mant-ADP), to the monomeric A subunit also supported the rapid nucleotide binding to the A subunit.     

V-Type H+-ATPase/synthase from a thermophilic eubacterium, Thermus thermophilus. Subunit structure and operon

V-type ATPase (V(o)V(1)) capable of ATP-driven H(+) pumping and of H(+) gradient driven ATP synthesis was isolated from a thermophilic eubacterium, Thermus thermophilus. When the enzyme was analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate, it showed eight polypeptide bands of which four were subunits of V(1). We also isolated the V(o)V(1) operon, containing nine genes in the order of atpG-I-L-E-X-F-A-B-D, which encoded proteins with molecular sizes of 13, 43, 10, 20, 35, 11, 64, 53, and 25 kDa, respectively. The last four genes were identified as those for V(1) subunits; atpA, B, D, and F encoded the A, B, gamma, and delta subunits, respectively. The first five genes, atpG-atpX, were identified as genes for the V(o) subunits. The product of atpL, the proteolipid subunit, lacked a 19-amino acid presequence and, unlike V-type ATPases, contained two membrane-spanning domains rather than four. The hydrophobic 43-kDa product of atpI is the smallest member so far found of the eukaryotic 100-kDa subunit family. Its electrophoretic band overlapped with the band of the A subunit. Therefore, all the gene products were found in our purified V(o)V(1). We isolated the A(3)B(3) subcomplex reconstituted from the isolated subunits and the A(3)B(3)gamma subcomplex from subunit-expressing Escherichia coli. Electron microscopic observation of these subcomplexes revealed that the gamma subunit of V(1) filled the central cavity of A(3)B(3) and might be central subunit, similar to the gamma subunit of F(1)-ATPase.     

Subunit arrangement in V-ATPase from Thermus thermophilus

The V0V1-ATPase of Thermus thermophilus catalyzes ATP synthesis coupled with proton translocation. It consists of an ATPase-active V1 part (ABDF) and a proton channel V0 part (CLEGI), but the arrangement of each subunit is still largely unknown. Here we found that acid treatment of V0V1-ATPase induced its dissociation into two subcomplexes, one with subunit composition ABDFCL and the other with EGI. Exposure of the isolated V0 to acid or 8 m urea also produced two subcomplexes, EGI and CL. Thus, the C subunit (homologue of d subunit, yeast Vma6p) associates with the L subunit ring tightly, and I (homologue of 100-kDa subunit, yeast Vph1p), E, and G subunits constitute a stable complex. Based on these observations and our recent demonstration that D, F, and L subunits rotate relative to A3B3 (Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315; Yokoyama, K., Nakano, M., Imamura, H., Yoshida, M., and Tamakoshi, M. (2003) J. Biol. Chem. 278, 24255-24258), we propose that C, D, F, and L subunits constitute the central rotor shaft and A, B, E, G, and I subunits comprise the surrounding stator apparatus in the V0V1-ATPase.     

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.     

Reassembly of a large multisubunit protein promoted by nonprotein factors. Effects of calcium and glycerol on the association of extracellular hemoglobin

The effects of cations and glycerol on the dissociation induced by pressure and on the reassembly of Glossoscolex paulistus hemoglobin were examined by light scattering, gel filtration, and electron microscopy. Calcium stabilized the quaternary structure of the hemoglobin against pressure dissociation. In the presence of 50 mM Ca2+, the half-dissociation pressure (p 1/2) increased by 400 bar, which corresponds to an average stabilization of -0.62 kcal/mol of dissociating subunit. Calcium also promoted a large increase in the yield of recovery of fully assembled hemoglobin at the expense of the partially dissociated (one-twelfth subunit) and fully dissociated forms. Glycerol protected the hemoglobin from pressure dissociation, increasing the half-dissociation pressure (p 1/2) and promoted an increase in the yield of recovery of fully assembled hemoglobin by about 40%. Addition of calcium after return to atmospheric pressure increased recovery of the fully associated form only in a long time scale (many days). The existence of time-dependent changes in the conformation of the dissociated subunits is suggested to explain the partial association to one-twelfth subaggregates (drifted forms) that lack the ability to reassemble to native hemoglobin. The promotion of reassembly by nonprotein factors (calcium and glycerol) is suggested to occur by preventing the formation of wrong intermediate forms (drifted one-twelfth subunits).     

A 100 kDa polypeptide associates with the V0 membrane sector but not with the active oat vacuolar H(+)-ATPase, suggesting a role in assembly

The vacuolar H(+)-ATPase (V-ATPase) is responsible for acidifying endomembrane compartments in eukaryotic cells. Although a 100 kDa subunit is common to many V-ATPases, it is not detected in a purified and active pump from oat (Ward J.M. and Sze H. (1992) Plant Physiol. 99, 925-931). A 100 kDa subunit of the yeast V-ATPase is encoded by VPH1. Immunostaining revealed a Vph1p-related polypeptide in oat membranes, thus the role of this polypeptide was investigated. Membrane proteins were detergent-solubilized and size-fractionated, and V-ATPase subunits were identified by immunostaining. A 100 kDa polypeptide was not associated with the fully assembled ATPase; however, it was part of an approximately 250 kDa V0 complex including subunits of 36 and 16 kDa. Immunostaining with an affinity-purified antibody against the oat 100 kDa protein confirmed that the polypeptide was part of a 250 kDa complex and that it had not degraded in the approximately 670 kDa holoenzyme. Co-immunoprecipitation with a monoclonal antibody against A subunit indicated that peripheral subunits exist as assembled V1 subcomplexes in the cytosol. The free V1 subcomplex became attached to the detergent-solubilized V0 sector after mixing, as subunits of both sectors were co-precipitated by an antibody against subunit A. The absence of this polypeptide from the active enzyme suggests that, unlike the yeast Vph1p, the 100 kDa polypeptide in oat is not required for activity. Its association with the free Vo subcomplex would support a role of this protein in V-ATPase assembly and perhaps in sorting.     

Electron-microscopic structure of the V-ATPase from mung bean

The vacuolar H⁺-ATPase from mung bean (Vigna radiata L. cv. Wilczek) was purified to homogeneity. The purified complex contained all the reported subunits from mung bean, but also included a 40-kDa subunit, corresponding to the membrane-associated subunit d, which has not previously been observed. The structure of the V-ATPase from mung bean was studied by electron microscopy of negatively stained samples. An analysis of over 6,000 single-particle images obtained by electron microscopy of the purified complex revealed that the complex, similar to other V-ATPases, is organized into two major domains V₁ and V_o with overall dimensions of 25 nm×13.7 nm and a stalk region connecting the V₁ and V_o domains. Several individual areas of protein density were observed in the stalk region, indicating its complexity. The projections clearly showed that the complex contained one central stalk and at least two peripheral stalks. Subcomplexes containing subunits A, B and E, dissociated from the tonoplast membrane by KI, were purified. The structure of the subcomplex was also studied by electron microscopy followed by single-molecule analysis of 13,000 projections. Our preliminary results reveal an area of high protein density at the bottom of the subcomplex immediately below the cavity formed by the A and B subunits, indicating the position of subunit E.Abbreviations MSA Multivariate statistical analysis - 2D, 3D Two-, three-dimensional - V-ATPase Vacuolar H⁺-ATPase     

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.     

ATP synthase complex from bovine heart mitochondria. Subunit arrangement as revealed by nearest neighbor analysis and susceptibility to trypsin

The nearest neighbor relationships of bovine mitochondrial H(+)-ATPase subunits were investigated by the chemical cross-linking approach using the homobifunctional cleavable reagents dithiobis(succinimidyl propionate) and disuccinimidyl tartrate. Cross-linked proteins were resolved by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Individual subunits were detected by silver staining or by Western blotting and staining with subunit-specific antisera. Products larger than 80,000 daltons were not analyzed. Interactions between F1 subunits included cross-links between gamma and delta as well as gamma and epsilon subunits. Among F0 subunit interactions were observed cross-links of (i) coupling factor 6 (F6) with 8-, 20-, and 24-kDa proteins, (ii) oligomycin sensitivity-conferring protein (OSCP) with 24-kDa protein, and (iii) 20-kDa protein with 24-kDa protein. In addition, several cross-links among subunits involving F1 and F0 sectors were detected. These included cross-links between F6 and alpha, F6 and gamma, OSCP and alpha/beta, and 24-kDa protein and alpha/beta. Thus, OSCP, F6, and the 24-kDa protein were found to form cross-links with both F1 and F0 subunits. The surface accessibility of F0 subunits was investigated by subjecting aliquots of F0 to trypsin treatment. Our data demonstrated that the rate of degradation was in the order OSCP greater than 24-kDa protein greater than or equal to F6 greater than subunit 6. The degradation of subunits of F0 was prevented in intact or reconstituted F1-F0. Based on our present and previously published observations, a model of H(+)-ATPase has been proposed wherein OSCP, F6, and the 24-kDa protein are placed in the stalk region and the alpha and beta subunits of F1-ATPase have been extended down to the membrane surface to enclose the stalk segment.     

Vma9p (subunit e) is an integral membrane V0 subunit of the yeast V-ATPase

The Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) is composed of 14 subunits distributed between a peripheral V1 subcomplex and an integral membrane V0 subcomplex. Genome-wide screens have led to the identification of the newest yeast V-ATPase subunit, Vma9p. Vma9p (subunit e) is a small hydrophobic protein that is conserved from fungi to animals. We demonstrate that disruption of yeast VMA9 results in the failure of V1 and V0 V-ATPase subunits to assemble onto the vacuole and in decreased levels of the subunit a isoforms Vph1p and Stv1p. We also show that Vma9p is an integral membrane protein, synthesized and inserted into the endoplasmic reticulum (ER), which then localizes to the limiting membrane of the vacuole. All V0 subunits and V-ATPase assembly factors are required for Vma9p to efficiently exit the ER. In the ER, Vma9p and the V0 subunits interact with the V-ATPase assembly factor Vma21p. Interestingly, the association of Vma9p with the V0-Vma21p assembly complex is disrupted with the loss of any single V0 subunit. Similarly, Vma9p is required for V0 subunits Vph1p and Vma6p to associate with the V0-Vma21p complex. In contrast, the proteolipids associate with Vma21p even in the absence of Vma9p. These results demonstrate that Vma9p is an integral membrane subunit of the yeast V-ATPase V0 subcomplex and suggest a model for the arrangement of polypeptides within the V0 subcomplex.     

Chromosomal localization of three vacuolar-H+ -ATPase 16 kDa subunit (ATP6V0C) genes in the murine genome

Vacuolar-H(+)-ATPase (V-H-ATPase) is a large multimeric protein composed of at least 12 distinct subunits. The 16-kDa hydrophobic proteolipid subunit (ATP6V0C; ATPase, H(+ )transporting, lysosomal 16 kDa, V0 subunit C) plays a central role in H(+) transport across cellular membranes. We have mapped three ATP6V0C genes (Atp6v0c, Atp6v0c-ps1 and Atp6voc-ps2) in the murine genome. Atp6v0c-ps1 and Atp6v0c-ps2 map to Chromosomes 7 and 6, respectively. Atp6v0c maps to Chromosome 17, closely linked to the Tsc2 locus and D17Mit55. This region of Chromosome 17 in mouse is homologous with chromosome 16 in human where the ATP6V0C gene is localized.     

Interaction of assembly protein AP-2 and its isolated subunits with clathrin

The clathrin assembly protein complex AP-2 is a multimeric subunit complex consisting of two 100-115-kDa subunits known as alpha and beta and 50- and 16-kDa subunits. The subunits have been dissociated and separated by ion-exchange chromatography in 7.5 M urea. Fractions highly enriched in either the alpha or beta subunit were obtained. The alpha fraction interacted with clathrin as evidenced by its ability to bind to preassembled clathrin cages. It also reacted with dissociated clathrin trimers under conditions that favor assembly of coat structures, but did not yield discrete clathrin polygonal lattices. The enriched beta fraction (containing small amounts of alpha) reacted with clathrin to yield intact coats with the incorporation of approximately equivalent amounts of alpha and beta subunits into the polymerized species; excess free beta subunit was unreactive. The AP-2 complex was also completely dissociated in a highly denaturing solvent, 6 M Gdn.HCl, and the constituent subunits of 100-115, 50, and 16 kDa were separated by gel filtration. In a coassembly assay with clathrin, the clathrin polymerizing activity was exclusively associated with the 100-kDa subunit fraction with stoichiometric incorporation of both alpha and beta subunits of 100 kDa into the polymerized coats, and with no requirement for 50- or 16-kDa subunits. These observations demonstrate that the assembly activity of the complex is associated with the alpha and beta subunits and suggest that both subunits, through independent interactions with clathrin, are required for expression of complete lattice assembly activity.     

Studies on (K+ + H+)-ATPase. VI. Determination on the molecular size by radiation inactivation analysis

(1) A (K+ + H+)-ATPase containing membrane fraction, isolated from pig gastric mucosa, has been further purified by means of zonal electrophoresis, leading to a 20% increase in specific activity and an increase in ratio of (K+ + H+)-ATPase to basal Mg2+-ATPase activity from 9 to 20. (2) The target size of (Na+ + K+)-ATPase, determined by radiation inactivation analysis, is 332 kDa, in excellent agreement with the earlier value of 327 kDa obtained from the subunit composition and subunit molecular weights. This shows that the Kepner-Macey factor of 6.4 X 10(11) is valid for membrane-bound ATPases. (3) The target size of (K+ + H+)-ATPase is 444 kDa, which, in connection with a subunit molecular weight of 110000, suggests a tetrameric assembly of the native enzyme. The ouabain-insensitive K+-stimulated p-nitrophenylphosphatase activity has a target size of 295 kDa. (4) In the presence of added Mg2+ the target sizes of the (K+ + H+)-ATPase and its phosphatase activity are decreased by about 15%, while that for the (Na+ + K+)-ATPase is not significantly changed. This observation is discussed in terms of a Mg2+-induced tightening of the subunits composing the (K+ + H+)-ATPase molecule.     

Cloning and sequencing of V-ATPase subunit d from mung bean and its function in passive proton transport

We have previously shown that vacuolar H+-ATPase subcomplex V_o from mung bean contains subunit d, however, its sequence and function were unknown. In the present study, we report the cloning and recombinant over expression of subunit d from mung bean in E. coli. To study the function of subunit d, two vacuolar H+-ATPase subcomplexes V_o from mung bean were purified-one containing subunits a and c(c’,c”) and the other containing subunits a, c(c’,c”) and d. After reconstitution of the purified V_o subcomplexes into liposomes, the proton translocation was studied. Our results show that the V_o subcomplex in the absence of subunit d is a passive proton channel, while the V_o subcomplex in the presence of the subunit d is not. Taken together, our data supports the conclusion that the subunit d of the plant vacuolar H⁺-ATPase from mung bean is positioned at the central stalk and involved in the proton translocation across the tonoplast membrane.     

H+-translocating ATPase in Golgi apparatus. Characterization as vacuolar H+-ATPase and its subunit structures

Golgi apparatus was prepared from rat liver, and enzymatic properties and the subunit structure of the H+-ATPase were characterized. GTP (and also ITP) was found to drive H+-transport with about 20% of the initial velocity as that of ATP. Bafilomycin, a specific inhibitor for vacuolar H+-ATPase, inhibited the activity at 2.5 nM. The H+-ATPase was completely inhibited in the cold in the presence of MgATP (5 mM) and NaNO3 (0.1 M). The cold inactivation of the H+-ATPase resulted in release of a set of polypeptides from Golgi membrane, with molecular masses almost identical to that of the hydrophilic sector of chromaffin granule H+-ATPase (72, 57, 41, 34, and 33 kDa). Three of these polypeptides (72, 57, and 34 kDa), cross-reacted with antibodies against the corresponding subunits of the chromaffin granule H+-ATPase. A counterpart of the 39-kDa hydrophobic component of chromaffin granule H+-ATPase was identified in the membrane, but no 115-kDa component was found. Hence, the Golgi H+-ATPase shows typical features of vacuolar H+-ATPase, in relatively low substrate specificity, its response to inhibitors, inactivation by cold treatment in the presence of MgATP, and subunit composition judged by antibody cross-reactivity.     

The transmembrane domain of subunit b of the Escherichia coli F1F(O) ATP synthase is sufficient for H(+)-translocating activity together with subunits a and c.

Subunit b is indispensable for the formation of a functional H(+)-translocating F(O) complex both in vivo and in vitro. Whereas the very C-terminus of subunit b interacts with F(1) and plays a crucial role in enzyme assembly, the C-terminal region is also considered to be necessary for proper reconstitution of F(O) into liposomes. Here, we show that a synthetic peptide, residues 1-34 of subunit b (b(1-34)) [Dmitriev, O., Jones, P.C., Jiang, W. & Fillingame, R.H. (1999) J. Biol. Chem.274, 15598-15604], corresponding to the membrane domain of subunit b was sufficient in forming an active F(O) complex when coreconstituted with purified ac subcomplex. H(+) translocation was shown to be sensitive to the specific inhibitor N,N'-dicyclohexylcarbodiimide, and the resulting F(O) complexes were deficient in binding of isolated F(1). This demonstrates that only the membrane part of subunit b is sufficient, as well as necessary, for H(+) translocation across the membrane, whereas the binding of F(1) to F(O) is mainly triggered by C-terminal residues beyond Glu34 in subunit b. Comparison of the data with former reconstitution experiments additionally indicated that parts of the hydrophilic portion of the subunit b dimer are not involved in the process of ion translocation itself, but might organize subunits a and c in F(O) assembly. Furthermore, the data obtained functionally support the monomeric NMR structure of the synthetic b(1-34).     

In vitro reassembly of active large ribosomal subunits of the halophilic archaebacterium Haloferax mediterranei.

The large ribosomal subunits of the halophilic archaebacterium Haloferax mediterranei have been reconstituted in vitro from the dissociated RNA and protein components. Efficient reassembly of particles fully active in poly(U)-directed polyphenylalanine synthesis requires a 2-h incubation at 42 degrees C in the presence of no less than 2.5 M concentrations of monovalent cations and of 60 mM magnesium. K+ and NH4+ ions are equally effective in promoting subunit reconstitution; however, maximal efficiency is attained when they are combined in a 1:2 molar ratio. The reassembly process requires no heat activation step, as under the appropriate ionic conditions it takes place spontaneously within the temperature range optimal for growth of H. mediterranei cells (40-45 degrees C).