ATPase Mutants Research Articles

An NH<sub>2</sub>-terminal deleted plasma membrane H<sup>+</sup>-ATPase is a dominant negative mutant and is sequestered in endoplasmic reticulum derived structures

The NH2-terminus of the plasma membrane H+-ATPase is one of the least conserved segments of this protein among fungi. We constructed and expressed a mutant H+-ATPase from Saccharomyces cerevisiae deleted at an internal peptide within the cytoplasmic NH2-terminus (D44-F116). When the enzyme was subjected to limited trypsinolysis it was digested more rapidly than wild type H+-ATPase. Membrane fractionation experiments and immunofluorescence microscopy, using antibodies against H+-ATPase showed that the mutant ATPase is retained in the endoplasmic reticulum. The pattern observed in the immunofluorescence microscopy resembled structures similar to Russell bodies (modifications of the endoplasmic reticulum membranes) recently described in yeast. When the wild type H+-ATPase was co-expressed with the mutant, wild type H+-ATPase was also retained in the endoplasmic reticulum. Co-expression of both ATPases in a wild type yeast strain was lethal, demonstrating that this is a dominant negative mutant.

Defects in the enterohepatic circulation of bile acids

ILE ACIDS serve at least two physiological roles, both of which are dependent on their enterohepatic circulation. Bile acid flux is the major driving force of bile production and they are essential for the normal absorption of fats from the gastrointestinal tract. Bile is a mixture of waste products, notably bilirubin and other conjugates, along with bile acids, lipids and inorganic ions, most of which are reabsorbed. Both functions now seem equally important. Whilst bile acids are an essential component of bile they are themselves toxic. The main function of other constituents, notably phospholipids and cholesterol, is to protect against the harmful effects of bile acids, through the formation of mixed micelles. The complete cycle of enterohepatic circulation of bile acids involves transport across two epithelia, as well as passage through the gut and portal blood. Only about 2% of the bile acids excreted daily into the GI tract are lost in the stool. A single transporter appears be responsible for this conservation through uptake in the distal ileum. The apical sodium-dependent bile salt transporter (ASBT) (1,2) is a transmembrane protein similar to the sodiumdependent bile acid transporter found in hepatocytes (NTCP) (3), which is discussed below. The gene is located on chromosome 13q33 and has the symbol SLClOA2 (4). The protein, when expressed in vitro has Ki values in the range of 3.3 PM (chenodeoxycholate) to 41.5 PM (cholate) for bile acids but very high Ki values for a number of other organic anions (5). This contrasts with NTCP, which appears to have a much broader specificity. Recessive, loss of function, mutations in the ASBT gene have been found in patients with primary bile acid malabsorption (4). Bile acids are removed from the portal circulation by hepatocytes. The major bile acid transporter in the basolateral membrane of hepatocytes is the sodium taurocholate co-transporting polypeptide (NTCP) (3). The organic anion transporting polypeptides are capable of transporting bile acids, along with many amphipathic compounds. Whether OATP plays a physiologically significant role in bile acid transport in not clear. Bile acid export from hepatocyes operates against a steep concentration gradient. The biliary bile acid concentration is at least lOOO-fold higher than that in hepatocytes. It is not surprising therefore that the transport of bile acids across this membrane has been shown to be an energy-dependent process. The major transporter is called the bile salt export pump and is encoded by ABCBll (previously called BSEP or SPGP). When the rat cDNA was expressed in vitro, the protein proved to be an effective ATP-dependent bile acid transporter with a Km for taurocholate of -5 ,uM (6). The protein has therefore been named the bile salt export pump. Mutations in the human gene have been found in patients with progressive familial intrahepatic cholestasis with normal levels of serum yGT (7). In those where it has been measured, they also have low levels of biliary bile acids; this correlates well with the low yGT. A similar phenotype has been found in patients with mutations in a P-type ATPase, also located in the hepatocyte canalicular membrane (8). The protein may well be acting as an aminophospholipid flipase. However, it is not clear how mutations in this gene lead to an interruption in the efflux of bile acids from hepatocytes. Most of the major bile acid transporter genes have now been cloned. The intracellular transport of bile acids is less well understood. Recessive mutations have so far been found in three transporter genes, though

Significance of lysine/glycine cluster structure in gastric H+,K+-ATPase.

Gastric H+,K+-ATPase consists of alpha- and beta-subunits. The catalytic alpha-subunit contains a very unique structure consisting of lysine and glycine clusters, KKK(or KKKK)AG(G/R)GGGK-(K/R)K, in the amino-terminal cytoplasmic region. This structure is well conserved in all gastric H+,K+-ATPases from different animal species, and was postulated to be the site controlling the access of cations (or proton) to its binding site. In this report, we studied the role of this unique structure by expressing several H+,K+-ATPase mutants of the alpha-subunit together with the wild-type beta-subunit in HEK-293 cells. Even after replacing all the positively-charged amino acid residues (six lysines and one arginine) in the cluster with alanine or removing all the glycine residues in the cluster, the mutants preserved the H+,K+-ATPase activity, and showed similar affinity for ATP and K+ as well as similar pH profiles as those of wild-type H+,K+-ATPase, indicating that the cluster is not indispensable for H+,K+-ATPase activity and not directly involved in determination of the affinity for cation (proton).

Mapping the role of active site residues for transducing an ATP-induced conformational change in the bovine 70-kDa heat shock cognate protein.

ATP binding induces a conformational change in 70-kDa heat shock proteins (Hsp70s) that facilitates release of bound polypeptides. Using the bovine heat shock cognate protein (Hsc70) as a representative of the Hsp70 family, we have characterized the effect of mutations on the coupling between ATP binding and the nucleotide-induced conformational change. Steady-state solution small-angle X-ray scattering and kinetic fluorescence measurements on a 60-kDa fragment of Hsc70 show that point mutations K71M, E175S, D199S, and D206S in the nucleotide binding cleft impair the ability of ATP to induce a conformational change. A secondary mutation in the peptide binding domain, E543K, "rescues" the ATP-induced transition for three of these mutations (E175S/E543K, D199S/E543K, and D206S/E543K) but not for K71M/E543K. Analysis of kinetics of the ATPase cycle confirm that these effects do not result from unexpectedly rapid ATP hydrolysis or slow ATP binding. Crystallographic structures of E175S, D199S, and D206S mutant ATPase fragment proteins show that the mutations do not perturb the tertiary structure of the protein but do significantly alter the protein-ligand interactions, due in part to an apparent charge compensation effect whereby mutating a (probably) negatively charged carboxyl group to a neutral serine displaces a K+ ion from the nucleotide binding cleft in two out of three cases (E175S and D199S but not D206S).

Stress factors acting at the level of the plasma membrane induce transcription via the stress response element (STRE) of the yeast Saccharomyces cerevisiae.

A variety of stress factors induces transcription via the stress response element (STRE) present in control regions of a number of genes of the yeast Saccharomyces cerevisiae. Induction of transcription involves nuclear translocation of the STRE-binding transcription activators Msn2p and Msn4p. The primary cellular events triggering this translocation are presently not well understood. In this investigation, we have observed that a number of factors acting at the level of the yeast plasma membrane, including the antifungal agent nystatin, the steroidal alkaloid tomatine, benzyl alcohol, a number of detergents and the plasma membrane H+-ATPase inhibitor diethylstilbestrol or mutations in the PMA1 gene encoding the plasma membrane ATPase, induce Msn2p nuclear accumulation and STRE-dependent transcription. At least some of the stress factors acting via STREs cause an increase in plasma membrane permeability, leading to a decrease in membrane potential, which might be a primary cellular stress signal. A decrease in internal pH triggered by permeabilization of the plasma membrane or a change in cAMP levels are at least not obligatory factors in intracellular stress signal transduction. The signal transduction pathway transmitting the signal generated at the plasma membrane to Msn2p is still unknown.

C-MYC interacts with INI1/hSNF5 and requires the SWI/SNF complex for transactivation function.

Chromatin organization plays a key role in the regulation of gene expression. The evolutionarily conserved SWI/SNF complex is one of several multiprotein complexes that activate transcription by remodelling chromatin in an ATP-dependent manner. SWI2/SNF2 is an ATPase whose homologues, BRG1 and hBRM, mediate cell-cycle arrest; the SNF5 homologue, INI1/hSNF5, appears to be a tumour suppressor. A search for INI1-interacting proteins using the two-hybrid system led to the isolation of c-MYC, a transactivator. The c-MYC-INI1 interaction was observed both in vitro and in vivo. The c-MYC basic helix-loop-helix (bHLH) and leucine zipper (Zip) domains and the INI1 repeat 1 (Rpt1) region were required for this interaction. c-MYC-mediated transactivation was inhibited by a deletion fragment of INI1 and the ATPase mutant of BRG1/hSNF2 in a dominant-negative manner contingent upon the presence of the c-MYC bHLH-Zip domain. Our results suggest that the SWI/SNF complex is necessary for c-MYC-mediated transactivation and that the c-MYC-INI1 interaction helps recruit the complex.

Oligomycin Induces a Decrease in the Cellular Content of a Pathogenic Mutation in the Human Mitochondrial ATPase 6 Gene

A T --> G mutation at position 8993 in human mitochondrial DNA is associated with the syndrome neuropathy, ataxia, and retinitis pigmentosa and with a maternally inherited form of Leigh's syndrome. The mutation substitutes an arginine for a leucine at amino acid position 156 in ATPase 6, a component of the F0 portion of the mitochondrial ATP synthase complex. Fibroblasts harboring high levels of the T8993G mutation have decreased ATP synthesis activity, but do not display any growth defect under standard culture conditions. Combining the notions that cells with respiratory chain defects grow poorly in medium containing galactose as the major carbon source, and that resistance to oligomycin, a mitochondrial inhibitor, is associated with mutations in the ATPase 6 gene in the same transmembrane domain where the T8993G amino acid substitution is located, we created selective culture conditions using galactose and oligomycin that elicited a pathological phenotype in T8993G cells and that allowed for the rapid selection of wild-type over T8993G mutant cells. We then generated cytoplasmic hybrid clones containing heteroplasmic levels of the T8993G mutation, and showed that selection in galactose-oligomycin caused a significant increase in the fraction of wild-type molecules (from 16 to 28%) in these cells.

The in vivo association of BiP with newly synthesized proteins is dependent on the rate and stability of folding and not simply on the presence of sequences that can bind to BiP.

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine lambdaI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as "kinetic traps." Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.

Both Lobes of the Soluble Receptor of the Periplasmic Histidine Permease, an ABC Transporter (Traffic ATPase), Interact with the Membrane-bound Complex: EFFECT OF DIFFERENT LIGANDS AND CONSEQUENCES FOR THE MECHANISM OF ACTION

The histidine permease of Salmonella typhimurium is an ABC transporter (traffic ATPase). The liganded soluble receptor, the histidine-binding protein HisJ, interacts with the membrane-bound complex HisQMP2 and stimulates its ATPase activity, which results in histidine translocation. In this study, we utilized HisJ proteins with mutations in either of the two lobes and wild type HisJ liganded with different substrates to show that each lobe carries an interaction site and that both lobes are involved in inducing (stimulating) the ATPase activity. We suggest that the spatial relationship between the lobes is one of the factors recognized by the membrane-bound complex in dictating the efficiency of the induction signal and of translocation. Several of the key residues involved have been identified. In addition, using constitutive ATPase mutants, we show that the binding protein provides some additional essential function(s) in translocation that is independent of the stimulation of ATP hydrolysis, and one possible mechanism is proposed, which includes the notion that liganded HisJ has different optimal conformations for signaling and for translocation.

The Chaperone BiP/GRP78 Binds to Amyloid Precursor Protein and Decreases Aβ40 and Aβ42 Secretion

Recent studies of cellular amyloid precursor protein (APP) metabolism demonstrate a beta-/gamma-secretase pathway resident to the endoplasmic reticulum (ER)/Golgi resulting in intracellular generation of soluble APP (APPsbeta) and Abeta42 peptide. Thus, these intracellular compartments may be key sites of amyloidogenic APP metabolism and Alzheimer's disease pathogenesis. We hypothesized that the ER chaperone immunoglobulin binding protein (BiP/GRP78) binds to and facilitates correct folding of nascent APP. Metabolic labeling and immunoprecipitation of transiently transfected human embryonic kidney 293 cells demonstrated co-precipitation of APP with GRP78, revealing their transient interaction in the ER. Maturation of cellular APP was impaired by this interaction. Furthermore, the levels of APPs, Abeta40, and Abeta42 recovered in conditioned medium were lower compared with cells transfected with APP alone. Co-expression with APP of GRP78 T37G, an ATPase mutant, almost completely blocked cellular APP maturation as well as recovery of APPs, Abeta40, and Abeta42 in conditioned medium. The inhibitory effects of GRP78 and GRP78 T37G on Abeta40 and Abeta42 secretion were magnified by co-expression with the Swedish mutation of APP (K670N/M671L). Collectively, these data suggest a transient and direct interaction of GRP78 with APP in the ER that modulates intracellular APP maturation and processing and may facilitate its correct folding.

Phosphorylation Region of the Yeast Plasma-membrane H+-ATPase: ROLE IN PROTEIN FOLDING AND BIOGENESIS

Mutations at the phosphorylation site (Asp-378) of the yeast plasma-membrane H+-ATPase have been shown previously to cause misfolding of the ATPase, preventing normal movement along the secretory pathway; Asp-378 mutations also block the biogenesis of co-expressed wild-type ATPase and lead to a dominant lethal phenotype. To ask whether these defects are specific for Asp-378 or whether the phosphorylation region as a whole is involved, alanine-scanning mutagenesis has been carried out to examine the role of 11 conserved residues flanking Asp-378. In the sec6-4 expression system (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940-7949), the mutant ATPases displayed varying abilities to reach the secretory vesicles that deliver plasma-membrane proteins to the cell surface. Indirect immunofluorescence of intact cells also gave evidence for a spectrum of behavior, ranging from mutant ATPases completely arrested (D378A, K379A, T380A, and T384A) or partially arrested in the endoplasmic reticulum to those that reached the plasma membrane in normal amounts (C376A, S377A, and G381A). Although the extent of ER retention varied among the mutants, the endoplasmic reticulum appeared to be the only secretory compartment in which the mutant ATPases accumulated. All of the mutant proteins that localized either partially or fully to the ER were also malfolded based on their abnormal sensitivity to trypsin. Among them, the severely affected mutants had a dominant lethal phenotype, and even the intermediate mutants caused a visible slowing of growth when co-expressed with wild-type ATPase. The effects on growth could be traced to the trapping of the wild-type enzyme with the mutant enzyme in the ER, as visualized by double label immunofluorescence. Taken together, the results indicate that the residues surrounding Asp-378 are critically important for ATPase maturation and transport to the cell surface.

Mutation to the glutamate in the fourth membrane segment of Na+,K+-ATPase and Ca2+-ATPase affects cation binding from both sides of the membrane and destabilizes the occluded enzyme forms.

The functional consequences of mutations Glu329 --> Gln in the Na+,K+-ATPase and Glu309 --> Asp in the sarco(endo)plasmic reticulum Ca2+-ATPase were analyzed and compared. Relative to the wild-type Na+,K+-ATPase, the Glu329 --> Gln mutant exhibited a 20-fold reduction in the apparent K+ affinity determined by titration of the rate of ATP hydrolysis at 50 microM ATP, and the rate of release of occluded K+ or Rb+ to the cytoplasmic side of the membrane was up to 30-fold enhanced by the mutation, as measured in kinetic studies of the phosphorylation by ATP of enzyme equilibrated with K+ or Rb+. The apparent affinity for extracellular K+ was 12-fold reduced by the Glu329 --> Gln mutation, as determined by K+ titration of the dephosphorylation. The maximum rate of phosphorylation by ATP of the Na+ form of the enzyme was reduced more than 2-fold by the mutation, but this effect could be counteracted by stabilizing Na+ occlusion with oligomycin. Similar studies on the Glu309 --> Asp mutant of the Ca2+-ATPase showed that the maximum rate of phosphorylation of the Ca2+ form was 8-9-fold reduced relative to that of the wild-type Ca2+-ATPase, and no Ca2+ occlusion could be detected in the mutant. Dephosphorylation of the phosphoenzyme intermediate formed with Pi was blocked in the Ca2+-ATPase mutant. The sensitivity to inhibition by thapsigargin, which binds selectively to the putative proton-occluded form of the Ca2+-ATPase, was reduced almost 300-fold in the mutant at neutral pH, but only 3-4-fold at pH 6.0. These data indicate that the mutations destabilize the occluded enzyme forms and interfere with cation binding from the extracytoplasmic side as well as with the gating process at the cytoplasmic entrance to the cation occlusion pocket.

Characterization of a Temperature-sensitive Yeast Vacuolar ATPase Mutant with Defects in Actin Distribution and Bud Morphology

The 27-kDa E subunit, encoded by the VMA4 gene, is a peripheral membrane subunit of the yeast vacuolar H+-ATPase. We have randomly mutagenized the VMA4 gene in order to examine the structure and function of the 27-kDa subunit. Cells lacking a functional VMA4 gene are unable to grow at pH > 7 or in elevated concentrations of CaCl2. Plasmid-borne, mutagenized vma4 genes were screened for failure to complement these phenotypes. Mutants producing Vma4 proteins detectable by immunoblot were selected; one (vma4-1(ts)) is temperature conditional, exhibiting the Vma- phenotype only at elevated temperature (37 degreesC). Sequencing revealed that a single point mutation, D145G, was responsible for the phenotypes of the vma4-1(ts) allele. The unassembled 27-kDa subunit made in the vma4-1(ts) cells is rapidly degraded, particularly at 37 degreesC, but can be protected from degradation by prior assembly into the V-ATPase complex. In purified vacuolar vesicles from the mutant cells, the peripheral subunits are localized to the vacuolar membrane at decreased levels and a comparably decreased level of ATPase activity (14% of the activity in wild-type vesicles) is observed. When vma4-1(ts) mutant cells are shifted to pH 7.5 medium at 37 degrees C, the cells become enlarged and exhibit multiple large buds, elongated buds, and other abnormal morphologies, together with delocalization of actin and chitin, within 4 h. These phenotypes suggest connections between the vacuolar ATPase, bud morphology, and cytokinesis that had not been recognized previously.

Direct Demonstration of Ca2+ Binding Defects in Sarco-Endoplasmic Reticulum Ca2+ ATPase Mutants Overexpressed in COS-1 Cells Transfected with Adenovirus Vectors

Single mutations of specific amino acids within the membrane-bound region of the sarco-endoplasmic reticulum Ca2+ (SERCA)-1 ATPase interfere with Ca2+ inhibition of ATPase phosphorylation by Pi (1), suggesting that these residues may be involved in complexation of two Ca2+ that are known to bind to the enzyme. However, direct measurements of Ca2+ binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficiency by means of recombinant adenovirus vectors, yielding sufficient expression of wild type and mutant SERCA-1 ATPase for measurements of Ca2+ binding to the microsomal fraction of the transfected cells. We find that in the presence of 20 microM Ca2+ and in the absence of ATP, the Glu771 --> Gln, Thr799 --> Ala, Asp800 --> Asn, and Glu908 --> Ala mutants exhibit negligible binding, indicating that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 are involved in interactions whose single disruption causes major changes in the highly cooperative "duplex" binding. Total loss of Ca2+ binding is accompanied by loss of Ca2+ inhibition of the Pi reaction. We also find that, at pH 7.0, the Glu309 --> Gln and the Asn796 --> Ala mutants bind approximately half as much Ca2+ as the wild type ATPase and do not interfere with Ca2+ inhibition of the Pi reaction. At pH 6.2, the Glu309 --> Gln mutant does not bind any Ca2+, and its phosphorylation by Pi is not inhibited by Ca2+. On the contrary, the Asn796 --> Ala mutant retains the behavior displayed at pH 7.0. This suggests that in the Glu309 --> Gln mutant, ionization of acidic functions in other amino acids (e.g. Glu771 and Asp800) occurs as the pH is shifted, thereby rendering Ca2+ binding possible. In the Asn796 --> Ala mutant, on the other hand, the Glu309 carboxylic function allows binding of inhibitory Ca2+ even at pH 6.2. In all cases mutational interference with the inhibition of the Pi reaction by Ca2+ can be overcome by raising the Ca2+ concentration to the mM range, consistent with a general effect of mutations on the affinity of the ATPase for Ca2+.

Structural replacement of active site monovalent cations by the epsilon-amino group of lysine in the ATPase fragment of bovine Hsc70.

We have assessed the ability of the epsilon-amino group of a non-native lysine chain to substitute for a monovalent cation in an enzyme active site. In the bovine Hsc70 ATPase fragment, mutation of cysteine 17 or aspartic acid 206 to lysine potentially allows the replacement of an active site potassium ion with the epsilon-amino nitrogen. We examined the ATP hydrolysis kinetics and crystal structures of isolated mutant ATPase domains. The introduced epsilon-amino nitrogen in the C17K mutant occupies a significantly different position than the potassium ion. The introduced epsilon-amino nitrogen in the D206K mutant occupies a position indistinguishable from that of the potassium in the wild-type structure. Each mutant retains <5% ATPase activity when compared to the wild type under physiological conditions (potassium buffer) although substrate binding is tighter, probably as a consequence of slower release. It is possible to construct a very good structural mimic of bound cation which suffices for substrate binding but not for catalytic activity.

Substitutions of Aspartate 378 in the Phosphorylation Domain of the Yeast PMA1 H+-ATPase Disrupt Protein Folding and Biogenesis

There is strong evidence that Asp-378 of the yeast PMA1 ATPase plays an essential role in ATP hydrolysis by forming a covalent beta-aspartyl phosphate reaction intermediate. In this study, Asp-378 was replaced by Asn, Ser, and Glu, and the mutant ATPases were expressed in a temperature-sensitive secretion-deficient strain (sec6-4) that allowed their properties to be examined. Although all three mutant proteins were produced at nearly normal levels and remained stable for at least 2 h at 37 degrees C, they failed to travel to the vesicles that serve as immediate precursors of the plasma membrane; instead, they became arrested at an earlier step of the secretory pathway. A closer look at the mutant proteins revealed that they were firmly inserted into the bilayer and were not released by washing with high salt, urea, or sodium carbonate (pH 11), treatments commonly used to strip nonintegral proteins from membranes. However, all three mutant ATPases were extremely sensitive to digestion by trypsin, pointing to a marked abnormality in protein folding. Furthermore, in contrast to the wild-type enzyme, the mutant ATPases could not be protected against trypsinolysis by ligands such as MgATP, MgADP, or inorganic orthovanadate. Thus, Asp-378 functions in an unexpectedly complex way during the acquisition of a mature structure by the yeast PMA1 ATPase.

A triple mutation in the a subunit of the Escherichia coli/Propionigenium modestum F1Fo ATPase hybrid causes a switch from Na+ stimulation to Na+ inhibition.

Previously we have shown that the Na+-translocating Escherichia coli (F1-delta)/Propionigenium modestum (Fo+delta) hybrid ATPase acquires a Na+-independent phenotype by the c subunit double mutation F84L, L87V that is reflected by Na+-independent growth of the mutant strain MPC8487 on succinate [Kaim, G., and Dimroth, P. (1995) J. Mol. Biol. 253, 726-738]. Here we describe a new class of mutants that were obtained by random mutagenesis and screening for Na+-independent growth on succinate. All six mutants of the new class contained four mutations in the a subunit (S89P, K220R, V264E, I278N). Results from site-specific mutagenesis revealed that the substitutions K220R, V264E, and I278N were sufficient to create the new phenotype. The resulting E. coli mutant strain MPA762 could only grow in the absence but not in the presence of Na+ ions on succinate minimal medium. This effect of Na+ ions on growth correlated with a Na+-specific inhibition of the mutant ATPase. The Ki for NaCl was 1. 5 mM at pH 6.5, similar to the Km for NaCl in activating the parent hybrid ATPase at this pH. On the other hand, activation by Li+ ions was retained in the new mutant ATPase. In the absence of Na+ or Li+, the mutant enzyme had the same pH optimum at pH 6.5 and twice the specific activity as the parent hybrid ATPase. In accordance with the kinetic data, the reconstituted mutant ATPase catalyzed H+ or Li+ transport but no Na+ transport. These results show for the first time that the coupling ion selectivity of F1Fo ATPases is determined by structural elements not only of the c subunit but also of the a subunit.

Mode of interaction of the single a subunit with the multimeric c subunits during the translocation of the coupling ions by F1F0 ATPases.

We have recently isolated a mutant (aK220R, aV264E, aI278N) of the Na+-translocating Escherichia coli/Propionigenium modestum ATPase hybrid with a Na+-inhibited growth phenotype on succinate. ATP hydrolysis by the reconstituted mutant ATPase was inhibited by external (N side) NaCl but not by internal (P side) NaCl. In contrast, LiCl activated the ATPase from the N side and inhibited it from the P side. A similar pattern of activation and inhibition was observed with NaCl and the ATPase from the parent strain PEF42. We conclude from these results that the binding sites for the coupling ions on the c subunits are freely accessible from the N side. Upon occupation of these sites, the ATPase becomes more active, provided that the ions can be further translocated to the P side through a channel of the a subunit. If by mutation of the a subunit this channel becomes impermeable for Na+, N side Na+ ions specifically inhibit the ATPase activity. These conclusions were corroborated by the observation that proton transport into proteoliposomes containing the mutant ATPase was abolished by N side but not by P side Na+ ions. In contrast, LiCl affected proton translocation from either side, similar to the sidedness effect of Na+ ions on H+ transport by the parent hybrid ATPase. If the ATPase carrying the mutated a subunit was incubated with 22NaCl and ATP, 1 mol 22Na+/mol enzyme was occluded. With the parent hybrid ATPase, 22Na+ occlusion was not observed. The occluded 22Na+ could be removed from its tight binding site by 20 mM LiCl, while incubation with 20 mM NaCl was without effect. Li+ but not Na+ is therefore apparently able to pass through the mutated a subunit and make the entrapped Na+ ions accessible again to the aqueous environment. These results suggest an ion translocation mechanism through F0 that in the ATP hydrolysis mode involves binding of the coupling ions from the cytoplasm to the multiple c subunits, ATP-driven rotation to bring a Na+, Li+, or H+-loaded c subunit into a contact site with the a subunit and release of the coupling ions through the a subunit channel to the periplasmic surface of the membrane.

Evidence for the involvement of vacuolar activity in metal(loid) tolerance: vacuolar-lacking and -defective mutants of Saccharomyces cerevisiae display higher sensitivity to chromate, tellurite and selenite.

The responses of Saccharomyces cerevisiae towards the oxyanions tellurite, selenite and chromate were investigated in order to establish the involvement of the yeast vacuole in their detoxification. Three mutants of S. cerevisiae with defective vacuolar morphology and function were used; mutant JSR180 delta 1 is devoid of any vacuolar-like structure while ScVatB and ScVatC are deficient in specific protein subunits of the vacuolar (V)-H(+)-ATPase. All the mutant strains showed increased sensitivity to tellurite and chromate compared to their parental strains. Such sensitivity of the mutants was associated with increased accumulation of tellurium and chromium. These results indicate that accumulation of both tellurium and chromium occurred mainly in the cytosolic compartment of the cell, with detoxification influenced by the presence of a functionally-active vacuole which may play a role in compartmentation as well as regulation of the cytosolic compartment for optimal expression of a detoxification mechanism, e.g. reduction. In contrast, the vacuolar-lacking mutant, JSR180 delta 1, and the defective V-H+ATPase mutant ScVatB displayed lower selenium accumulation than their parental strains. Additionally, the mutant strain ScVatB displayed a higher tolerance to selenite than the parental strain. This result suggests that accumulation of selenium occurs mainly in the vacuolar compartment of the cell with tolerance depending on the ability of the cytosolic component to reduce selenite to elemental selenium, which might, in turn, be related to activity of the V-H(+)-ATPase. These results are discussed in relation to vacuolar compartmentation and the significance of the vacuolar H(+)-ATPase in cytosolic homeostasis of H+ both of which may affect the accumulation, reduction, and tolerance to the tested metal(loids).

Isolation and characterization of a neomycin-resistant mutant of Methanobacterium thermoautotrophicum with a lesion in Na +-translocating ATPase (synthase)

A mutant of Methanobacterium thermoautotrophicum with a lesion in membrane Na +-translocating ATPase (synthase) was isolated. The total ATPase activity in permeabilized cells of this mutant was elevated three-fold as compared with the wild-type strain. In contrast to wild-type cells, mutant ATPase was neither inhibited by DCCD nor stimulated by Na + ions. The methane formation rate of the mutant cells at pH 7.5 under non-growing conditions was nearly twice that of the wild-type strain and was stimulated by sodium ions. On the other hand, the ATP synthesis driven by methanogenesis under the same conditions was lower that of the wild-type under the same conditions, and contrary to the wild-type was not stimulated by Na + ions. ATP synthesis driven by a potassium diffusion potential in the presence of sodium ions was markedly diminished in the mutant cells. The membrane potential values of the wild-type and the mutant cells in the presence of 10 mM NaCl at pH 7.0 were comparable at energized conditions (−223 mV and −230 mV respectively). The Mg 2+-dependent ATPase activity of the 10 5× g supernatant of broken cells from the mutant cells was 30% higher than in the wild-type. On the other hand, two bands with Mg 2+-dependent ATPase activity were identified by native PAGE in this fraction in both wild-type as well as in mutant. These data suggest that the binding of Na +-translocating ATPase (synthase) to the membrane spanning part is changed in the mutant strain.