Takato Noumi

Essential aspartic acid residues, Asp-133, Asp-163 and Asp-164, in the transmembrane helices of a Na+/H+ antiporter (NhaA) from Escherichia coli

The importance of negatively charged residues in transmembrane helices of many cation‐coupled transporters has been widely demonstrated. Four Asp residues were located in the putative transmembrane helices of the Escherichia coli Na+/H+ antiporter, NhaA. We replaced each of these Asp residues by Asn in plasmid encoded nhaA and expressed these constructs in an E. coli mutant defective in both nhaA and nhaB. Substitution of Asp‐65 or Asp‐282 (in the extramembrane region) had no effect on supporting the host mutant growth in the high NaCl‐ or LiCl‐containing medium, and these two mutants had normal Na+/H+ and Li+/H+ antiporter activities. In contrast, substitution of Asp‐133, Asp‐163 or Asp‐164 was detrimental to survival of the host mutant and impaired both N+/H+ and L+/H+ antiporter activities. These three Asp residues, conserved in the nhaA homologs from different species and which are located closely in the 3rd and 4th putative transmembrane helices, appear to play important roles in cation binding and transport.

TARGETED DISRUPTION OF THE GENE ENCODING THE PROTEOLIPID SUBUNIT OF MOUSE VACUOLAR H+-ATPASE LEADS TO EARLY EMBRYONIC LETHALITY

Vacuolar H(+)-ATPase (V-ATPase) is responsible for acidification of intracellular compartments in eukaryotic cells. Its 16-kDa subunit (proteolipid, PL16) plays a central role in V-ATPase function, forming the principal channel via which protons are translocated. To elucidate physiological roles of V-ATPase in mammalian cell function and embryogenesis, we attempted to generate a PL16 null mutant mouse by gene-targeting. Mice heterozygous (PL16(+/-)) for the proteolipid mutation were intercrossed and their offspring were classified according to genotype. There were no homozygous (PL16(-/-)) pups among 69 neonates examined, but a few PL16(-/-) embryos were found during the pre-implantation stages of embryonic development, up to day 3.5 post-coitum. These results suggested that PL16 (and hence V-ATPase) may play an essential role in cell proliferation and viability during early embryogenesis. PL16(+/-) mice were indistinguishable from their wild-type littermates and displayed no discernible abnormalities, although the PL16 mRNA level in PL16(+/-) mice decreased to about one-half of wild-type levels.

Nucleotide sequence of the genes for F0 components of the proton-translocating ATPase from Escherichia coli: Prediction of the primary structure of F0 subunits

The 1763 nucleotide-long-DNA sequence of part of the gene cluster for the proton-translocating ATPase from E. coli was determined. The sequence covers the genes for the a and b subunits of F0 along with the intercistronic regions. In the region preceding the gene for the a subunit, a reading frame encompassing 127 amino acids was found. The primary structure of the a and b subunits were deduced and the properties of these proteins were predicted. Analysis of codon usage in these genes was made.

Mutants of Escherichia coli H+-ATPase defective in the δ subunit of F1 and the b subunit of F0

Summary Complete nucleotide sequence of the genes for subunits of the H+ ATPase of E. coli has been determined and several hybrid plasmids carrying various portions of these genes have been constructed. Genetic complementation and recombination tests of about forty mutants of E. coli defective in the ATPase were performed using these plasmids for identifying the locations of the mutations. Two mutants defective in the 8 subunit and a novel type of mutant defective in the b subunit of F0 were identified. The δ subunit mutants showed no proton conduction, suggesting that this subunit has an important role for the proton conduction. The ATPase of the b subunit mutant has a normal activity of proton channel portion, which phenotype is clearly different from that of mutants of the b subunit reported previously.

Intra- and intermolecular interactions of the catalytic domains of human CD45 protein tyrosine phosphatase

We have investigated protein‐protein interaction between distinct domains of the human CD45 cytoplasmic region using a yeast two‐hybrid system. Consequently, we have found that the spacer region between two tandem PTP domains specifically interacts with the membrane‐distal PTP domain (D2). This interaction is mediated by a stretch of amino acid residues in the carboxyl‐terminal half of the spacer region. Although the membrane proximal region does not directly interact with either of the two PTP domains, it appears to function in stabilizing the interaction between the spacer region and D2. We also demonstrate that the interaction between the spacer region and D2 might take place intramolecularly.

[60] Analysis of Escherichia coli mutants of the H+-transporting ATPase: Determination of altered site of the structural genes

Publisher Summary This chapter describes the analysis of Escherichia coli mutants of the H + -transporting ATPase for the determination of altered site of the structural genes. The chapter presents the procedures for location of a mutation, taking mutations in the β subunit of F 1 -ATPase as examples: (1) rapid mapping of a mutation site within a defined domain of a subunit by a genetic recombination test, (2) cloning of a mutation site, (3) detection of anomalous migration of DNA fragments carrying a mutation, and (4) determination of an altered nucleotide sequence. Several mutations within a defined portion of the cistron for the α, β, γ, or b subunits by genetic recombination tests with plasmids carrying various portions of each gene have been located. The chapter presents the results that not only support genetic results, but also eliminate tedious determination of the complete nucleotide sequence of a gene to find the single base change. Sequencing of only the portion of the gene carrying a mutation is sufficient for determination of the mutated base, even in the larger subunits α and β.

Escherichia coli mutants defective in the γ subunit of proton-translocating ATPase: Intracistronic mapping of the defective site and the biochemical properties of the mutants

Various hybrid plasmids carrying a portion of the gene for the gamma subunit of the H+-ATPase of Escherichia coli complemented five mutants defective in the enzyme in a genetic test, indicating that the mutants are defective in the gamma subunit. Since the nucleotide sequence of genomic DNA carried on the plasmids is known, the defective site(s) of the mutants could be located within the gene for the gamma subunit as follows: KF10 and NR70, KF1, and KF12 and KF13 have a mutation causing a defect(s) in amino acid residues 1 to 82, 83 to 167, and 168 to 287, respectively, of the gamma subunit. The biochemical properties of all these mutants except NR70 were analyzed in terms of proton permeability of the membranes and assembly of F1. Results suggested that KF1 and KF10 have defective F1 without at least the alpha and beta subunits on their membranes, whereas KF12 and KF13 have F1s of rather similar structure to that of the wild type. Attempts were made to purify F1 oF KF12 as a single complex. Although the F1 complex dissociated during purification, active alpha and beta subunits of KF12 were partially purified. On the basis of these biochemical and genetic results, it is suggested that structural alterations in the primary sequence of the gamma subunit corresponding to residues 1 to 167 cause more extensive defects in the assembly of F1 than alteration in the sequence of residues 168 to 287.

Insertions of transposable elements in the promoter proximal region of the gene cluster for Escherichia coli H+-ATPase: 8 base pair repeat generated by insertion of IS1

SummaryA plasmid pKY159 (Yamaguchi and Yamaguchi 1983) carrying a promoter proximal portion of the gene cluster of the proton-translocating ATPase (H+-ATPase) of Escherichia coli causes growth inhibition of wild-type cells. Insertion of a transposable element in this plasmid released this inhibitory effect. In analyzing this inhibitory effect, we determined the insertion points at the nucleotidesequence level of transposable elements on 30 independent derivatives of pKY159. Insertions of IS1, IS5, and λδ were found between the promoter and the gene for a possible component of 14,000 daltons of the H+-ATPase. Of 31 insertions, 26 were of IS1 and were located at the same site, indicating that this site is a hotspot for IS1 insertion and that IS1 insertion is much more frequent than that of IS5 of λδ in this region. Four different sites for IS1 insertion were found; in two of these an 8 base pair (bp) duplicate of the target sequence (AAAAACGT and AAACGTTG) was generated, while in the other two a 9 bp duplicate was found. In all cases in this study the nucleotide sequence of IS1 was the same as that of IS1-K. In the two cases with an 8 bp duplicate in different sites, a common 6 bp sequence (AAACGT) was found. These results suggested that generation of the 8 bp duplicate is related to the common sequence rather than a mutation in IS1 suggested by Iida et al. (1981) and also suggested that the essential length of the duplicate is 8 bp or less than 8 bp. A 6 bp sequence (GTGATG) homologous to the end portion of IS1 was found at the hotspot, but not at other sites, suggesting that this homology contributed to the high frequency of IS1 insertion. The direction of IS1 insertion at the hotspot was the same in 25 of 26 instances, suggesting that the direction of IS1 insertion is determined by the structure of the target and/or its nearby sequence.

Intracistronic mapping of the defective site and the biochemical properties of β subunit mutants of Escherichia coli H+-ATPase: Correlation of structural domains with functions of the β subunit

Sixteen mutants of Escherichia coli defective in H+-ATPase (proton-translocating ATPase) were tested for their ability to recombine with hybrid plasmids carrying various portions of the beta subunit cistron. Twelve mutations were mapped within the carboxyl half of the cistron corresponding to amino acid residues 279 to 459 (domain II), while four mutations were mapped within residues 17 to 278 (domain I). The biochemical properties of these mutants were analyzed in terms of the proton permeability of their membranes and the assembly properties of their F1F0 complex. The mutants were classified according to the properties into three types, I, II, and III. In 12 mutants of type I, proton conduction in membrane vesicles was blocked and little F1 was released from the membranes under conditions in which F1 could be released from wild-type membranes, suggesting that assembly of the F1F0 complex is structurally and functionally defective. F1 was partially purified with very low recovery from one of the type I mutants, KF16. ATPase activity was reconstituted from this F1 with the beta subunit of the wild type, confirming the genetic results. Only one mutant, KF38, was classified as type II. Its membranes were partially leaky to protons and its F1 was releasable, suggesting that the interaction of its F1 and F0 was unstable. Type III mutants, KF11 and KF43, had an F1F0 complex with very low activity, in which the structure of F1 was relatively similar to that of the wild type. F1 was purified as a single complex from KF43 in this study and from KF11 previously (H. Kanazawa, Y. Horiuchi, M. Takagi, Y. Ishino, and M. Futai (1980) J. Biochem. 88, 695-703). Reconstitution experiments in vitro showed that the F1s of both mutants were defective in the beta subunit. The properties of the altered F1 of KF43 differed from those of F1 of KF11, suggesting that the mutation sites of KF43 and KF11 were different. From the results of mapping mutation sites and the biochemical properties of the mutants, the correlation of structural domains with function of the beta subunit is discussed. Most type I and type II mutations except that of KF39 were mapped in domain II, while the type III mutations were mapped in domain I, suggesting that domain II is more important than domain I for the function of the beta subunit, especially in terms of proper assembly of the F1F0 complex.

Reconstitution of the F1-ATPase activity from purified α, β γ and δ or ϵ subunits with glutathione S-transferase fused at their amino termini

Systems for overexpression and purification of active α, β and γ subunits of Escherichia coli H+-ATPase were established. The α and β subunits recovered as soluble form were purified by hydroxyapatite column chromatography. Since the γ subunit was overexpressed as the insoluble form, this subunit was purified by polyacrylamide gel-electrophoresis containing sodium dodecyl sulfate. By subsequent denaturation of this subunit with guanidine hydrochloride and renaturation, the active γ subunit for reconstitution of the F1-ATPase activity with the purified α and β subunit was obtained. The δ and ϵ subunits which were fused to the carboxy terminus of glutathione S-transferase (GST) were overproduced and purified by affinity chromatography. These fused proteins (δ-GST and ϵ-GST) were incubated with the purified α, β and γ subunits and applied to affinity chromatography. The αβγδ-GST and αβγϵ-GST complex were eluted specifically by addition of glutathione and exhibited high and low ATPase activity, respectively, with a subunit stoichiometry similar to that in the native F1-ATPase, indicating that active complexes could be reconstituted with the fused proteins. These results suggested that the amino-terminal ends of the δ and ϵ subunits are not involved in formation of the active complex. The fused ϵ-GST bound the y subunit strongly, and the a subunit weakly. The δ-GST bound the γ subunit significantly, and the α and β subunits very weakly.