A point mutation within each of two ATP-binding motifs inactivates the functions of elongation factor 3

Yang, Hemiing; Hamada, Kenji; Terashima, Hiromichi; Izuta, Miho; Yamaguchi-Sihta, Emi; Kondoh, Osamu; Satoh, Hideo; Miyazaki, Motoichi; Arisawa, Mikio; Miyamoto, Chikara; Kitada, Koji

doi:10.1016/0167-4889(95)00179-4

Cited by 14 publications

(12 citation statements)

References 9 publications

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“…ATPase and reduced its intrinsic ATPase activity, leading to increased oxidative stress and increased sensitivity to respiratory inhibitors (15,16,(23)(24)(25)(26). However, the role of ATP/GDP-binding protein motif A (P-loop) in the virulence of brucellae is still unknown.…”

Section: Discussionmentioning

confidence: 99%

“…The looP gene has been identified previously in E. coli, Saccharomyces cerevisiae, and Porphyromonas gingivalis, and the motif is commonly found in many nucleotide-binding proteins, including ATPases, ATP synthases, kinases, elongation factors, and myosin (15,16,23). In fact, a mutation within the P-loop ATP-binding motif abolished most of the activity of the ribosome-activated (C) Sensitivity to acidic stress was determined after all strains were exposed for 3 h to TSB adjusted to pH 7.0, pH 4.4, or pH 3.5.…”

Section: Discussionmentioning

confidence: 99%

“…Mutants defective in cydC have a deficiency of cytochrome bd oxidase and an inability to exit from stationary phase and are hypersensitive to zinc and oxidative stress. However, the looP gene has not been fully characterized (15,16). The roles of the looP and cydC genes in the virulence of B. abortus are not well established.…”

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confidence: 99%

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Characterization and Protective Property of Brucella abortuscydCandlooPMutants

Truong¹,

Cho²,

Barate³

et al. 2014

Clin. Vaccine Immunol.

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bBrucella abortus readily multiplies in professional or nonprofessional phagocytes in vitro and is highly virulent in mice. Isogenic mutants of B. abortus biovar 1 strain IVKB9007 lacking the ATP/GDP-binding protein motif A (P-loop) (named looP; designated here the IVKB9007 looP::Tn5 mutant) and the ATP-binding/permease protein (cydC; designated here the IVKB9007 cydC::Tn5 mutant) were identified and characterized by transposon mutagenesis using the mini-Tn5Km2 transposon. Both mutants were found to be virtually incapable of intracellular replication in both murine macrophages (RAW264.7) and the HeLa cell line, and their virulence was significantly impaired in BALB/c mice. Respective complementation of the IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants restored their ability to survive in vitro and in vivo to a level comparable with that of the wild type. These findings indicate that the cydC and looP genes play important roles in the virulence of B. abortus. In addition, intraperitoneal immunization of mice with a dose of the live IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants provided a high degree of protection against challenge with pathogenic B. abortus strain 544. Both mutants should be evaluated further as a live attenuated vaccine against bovine brucellosis for their ability to stimulate a protective immune response. Brucella abortus, the causative agent of bovine brucellosis, causes abortion and reduced fertility in cattle and undulant fever in humans (1). Unlike other pathogenic bacteria, brucellae do not have classical virulence factors. A key aspect of the virulence of B. abortus is its ability to invade, survive, and proliferate within host phagocytic cells; it successfully bypasses the bactericidal activities of phagocytes and thereby establishes long-lasting chronic infections (2, 3). The B. abortus vaccine strains S19 and RB51 have been used worldwide for the prevention of brucellosis in cattle but have several drawbacks, including interference with diagnosis, residual virulence, and some pathogenicity for humans as well as cattle (4, 5). Therefore, understanding the virulence of B. abortus on a genetic level and the host response against B. abortus will provide important information for the development of a new vaccine for controlling brucellosis.Transposon mutagenesis is an extensively used approach for the identification of genes involved in the virulence of bacterial pathogens (6-8). To understand the sustained intracellular residence of brucellae in host cells, a transposon-based approach has been employed to identify genes in brucellae that are critically required for lipopolysaccharide biosynthesis, metabolic processes, stress responses, nutrient deprivation, and invasion of and survival in host cells. Mutations in these genes led to the attenuation of Brucella compared with the virulence of the parental strain; attenuation was recognized by the reduced intracellular survival and replication or rapid clearance of the mutants from both macrophages and mice (6-11). However, these atten...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Characterization and Protective Property of Brucella abortuscydCandlooPMutants

Truong¹,

Cho²,

Barate³

et al. 2014

Clin. Vaccine Immunol.

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“…These residues were selected because the corresponding residues in the Walker A motif in eEF3 are essential for binding to ATP and the ensuing generation of ribosomeactivated ATPase activity. Mutation of these residues in eEF3 abolishes its ribosome-dependent ATPase activity and ability to support poly(U)-directed polyphenylalanine synthesis and also impair cell growth (33). We expressed and purified recombinant wild type His 6 -ABC50 or the single and double mutated proteins in E. coli (pET28c-ABC50 WT, K304M, K626M, and K304M/K626M).…”

Section: Functional Nbds Are Not Required For the Association Of Abc5mentioning

confidence: 99%

ABC50 Promotes Translation Initiation in Mammalian Cells

Paytubi

Wang

Lam

et al. 2009

Journal of Biological Chemistry

120

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ABC50 is an ATP-binding cassette (ABC) protein, which, unlike most ABC proteins, does not possess membrane-spanning domains. ABC50 interacts with eukaryotic initiation factor 2 (eIF2), which plays a key role in translation initiation and its control. ABC50 binds to ribosomes, and this interaction requires both the N-terminal domain and at least one ABC domain. Knockdown of ABC50 by RNA interference impaired translation of both cap-dependent and -independent reporters, consistent with a positive role for ABC50 in the function of eIF2, which is required for both types of translation initiation. Mutation of the Walker box A or B motifs in both ABC regions of ABC50 yielded a mutant protein that exerted a dominant-interfering phenotype with respect to protein synthesis and translation initiation. Importantly, although dominant-interfering mutants of ABC50 impaired cap-dependent translation, translation driven by certain internal ribosome entry segments was not inhibited. ABC50 is located in the cytoplasm and nucleoplasm but not in the nucleolus. Thus, ABC50 is not likely to be directly involved in early ribosomal biogenesis, unlike some other ABC proteins. Taken together, the present data show that ABC50 plays a key role in translation initiation and has functions that are distinct from those of other non-membrane ABC proteins.

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“…The Walker C motif is the conserved LSGGQ sequence, the presence of which distinguishes the ABC proteins from other ATPases (14). Alterations of the conserved glycine and lysine residues within the Walker A of either ABC1 or -2 abolish the ATP hydrolytic activity of eEF3 in vitro and are lethal for growth in vivo (15). Interestingly, a temperature-sensitive (Ts Ϫ ) F650S point mutant in the intervening region of the two ABC cassettes also affects the catalytic ATPase activity of the protein, indicating that the linker region affects either ATP binding or its hydrolysis (16).…”

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confidence: 99%

Domain and Nucleotide Dependence of the Interaction between Saccharomyces cerevisiae Translation Elongation Factors 3 and 1A

Anand¹,

Balar²,

Ulloque³

et al. 2006

Journal of Biological Chemistry

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Eukaryotic translation elongation factor 3 (eEF3) is a fungalspecific ATPase proposed to catalyze the release of deacylatedtRNA from the ribosomal E-site. In addition, it has been shown to interact with the aminoacyl-tRNA binding GTPase elongation factor 1A (eEF1A), perhaps linking the E and A sites. Domain mapping demonstrates that amino acids 775-980 contain the eEF1A binding sites. Domain III of eEF1A, which is also involved in actin-related functions, is the site of eEF3 binding. The binding of eEF3 to eEF1A is enhanced by ADP, indicating the interaction is favored post-ATP hydrolysis but is not dependent on the eEF1A-bound nucleotide. A temperaturesensitive P915L mutant in the eEF1A binding site of eEF3 has reduced ATPase activity and affinity for eEF1A. These results support the model that upon ATP hydrolysis, eEF3 interacts with eEF1A to help catalyze the delivery of aminoacyl-tRNA at the A-site of the ribosome. The dynamics of when eEF3 interacts with eEF1A may be part of the signal for transition of the post to pre-translocational ribosomal state in yeast.The protein synthetic machinery is characterized by the interplay of different soluble factors in conjunction with ribosomes to translate the mRNA into the correct sequence of amino acids. The three phases of translation, initiation, elongation, and termination, are driven by factors that are highly conserved between yeast and metazoans (1). However, a major difference in elongation is the indispensability of eukaryotic elongation factor 3 (eEF3) 3 with yeast ribosomes (2, 3). eEF3 catalyzes an essential step in each elongation cycle by virtue of its ATPase activity. It has been proposed to act as an Exit-site (E-site) factor, facilitating the release of deacylated-tRNA and simultaneously impacting on the delivery of aminoacyl-tRNA (aa-tRNA) at the aminoacyl site (A-site) (4). Metazoan ribosomes have been reported to possess a compensatory intrinsic ATPase activity, although they differ kinetically from the fungal eEF3 (5). Escherichia coli, on the other hand, expresses the 911 amino acid RbbA protein that exhibits ATPase activity and is tightly associated with ribosomes (6, 7). Both pathogenic and non-pathogenic fungi have been reported to contain eEF3 (8 -10). In Saccharomyces cerevisiae, eEF3 is encoded by a single copy essential YEF3 gene. A paralog of the YEF3 gene, designated HEF3 or YEF3B, encodes an 84% identical protein but is not expressed during vegetative growth (11). However, expression of the HEF3 coding sequence under the YEF3 promoter produces a protein that has similar ATPase activity and ribosome binding properties to YEF3-encoded eEF3.eEF3 is a class 1 member of the ATP binding cassette (ABC) family of proteins. eEF3 possesses distinct motifs including the HEAT repeats on the N terminus, two nucleotide binding domains with tandemly arranged bipartite (ABC) cassettes in the middle, a conserved insertion in the intervening region of the Walker A and B motifs of ABC2, and a highly basic C terminus. HEAT (Huntington elongation factor 3, ...

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A point mutation within each of two ATP-binding motifs inactivates the functions of elongation factor 3

Cited by 14 publications

References 9 publications

Characterization and Protective Property of Brucella abortuscydCandlooPMutants

Characterization and Protective Property of Brucella abortuscydCandlooPMutants

ABC50 Promotes Translation Initiation in Mammalian Cells

Domain and Nucleotide Dependence of the Interaction between Saccharomyces cerevisiae Translation Elongation Factors 3 and 1A

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