Tu YH et al. (2018) Science "An evolutionarily conserved gene family encodes proton-selective ion channels."

Teng B, Chang RB, Ye W, Turner HN, Mulhall EM, Smith AD, Tu YH, Artiga DJ, Cooper AJ, Liman ER

[Science, 2018]

Ion channels form the basis for cellular electrical signaling. Despite the scores of genetically identified ion channels selective for other monatomic ions, only one type of proton-selective ion channel has been found in eukaryotic cells. By comparative transcriptome analysis of mouse taste receptor cells, we identified Otopetrin1 (OTOP1), a protein required for development of gravity-sensing otoconia in the vestibular system, as forming a proton-selective ion channel. We found that murine OTOP1 is enriched in acid-detecting taste receptor cells and is required for their zinc-sensitive proton conductance. Two related murine genes, <i>Otop2</i> and <i>Otop3</i>, and a <i>Drosophila</i> ortholog also encode proton channels. Evolutionary conservation of the gene family and its widespread tissue distribution suggest a broad role for proton channels in physiology and pathophysiology.

Ohtsuki T et al. (2002) Nucleic Acids Res "Characterization of the interaction between the nucleotide exchange factor EF-Ts from nematode mitochondria and elongation factor Tu."

Ohtsuki T, Sato A, Sakurai M, Watanabe K

[Nucleic Acids Res, 2002]

Caenorhabditis elegans mitochondria have two elongation factor (EF)-Tu species, denoted EF-Tu1 and EF-Tu2. Recombinant nematode EF-Ts purified from Escherichia coli bound both of these molecules and also stimulated the translational activity of EF-Tu, indicating that the nematode EF-Ts homolog is a functional EF-Ts protein of mitochondria. Complexes formed by the interaction of nematode EF-Ts with EF-Tu1 and EF-Tu2 could be detected by native gel electrophoresis and purified by gel filtration. Although the nematode mitochondrial (mt) EF-Tu molecules are extremely unstable and easily form aggregates, native gel electrophoresis and gel filtration analysis revealed that EF-Tu.EF-Ts complexes are significantly more soluble. This indicates that nematode EF-Ts can be used to stabilize homologous EF-Tu molecules for experimental purposes. The EF-Ts bound to two eubacterial EF-Tu species (E.coli and Thermus thermophilus). Although the EF-Ts did not bind to bovine mt EF-Tu, it could bind to a chimeric nematode-bovine EF-Tu molecule containing domains 1 and 2 from bovine mt EF-Tu. Thus, the nematode EF-Ts appears to have a broad specificity for EF-Tu molecules from different

Kjeldgaard M (2002) Structure "Another worm in translation."

Kjeldgaard M

[Structure, 2002]

Elongation factor EF-Tu is a key component in the translation step of protein synthesis, where it forms a complex with amino-acyl tRNA and delivers it to the ribosome. Until now, none of the known EF-Tu molecules have discriminated between the different species of tRNA, but now a new discovery sheds light on a curious EF-Tu homolog that binds just a single tRNA species.

Ohtsuki T et al. (2002) Nat Struct Biol "A unique serine-specific elongation factor Tu found in nematode mitochondria."

Watanabe K, Watanabe Y, Sato A, Ohtsuki T

[Nat Struct Biol, 2002]

The translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNAs to ribosomes by recognizing the tRNA acceptor and T stems. However, the unusual truncation observed in some animal mitochondrial tRNAs seems to prevent recognition by a canonical EF-Tu. For instance, nematode mitochondria contain tRNAs lacking a T or D arm. We recently found an atypical EF-Tu (EF-Tu1) specific for nematode mitochondrial tRNAs that lack the T arm. We have now discovered a second factor, EF-Tu2, which binds only to tRNAs that lack a D arm. EF-Tu2 seems unique in its amino acid specificity because it recognizes the aminoacyl moiety of seryl-tRNAs and the tRNA structure itself. Such EF-Tu evolution might explain tRNA structural divergence in animal mitochondria.

Ohtsuki T et al. (2001) J Biol Chem "An "elongated" translation elongation factor Tu for truncated tRNAs in nematode mitochondria."

Watanabe Y, Ueda T, Kita K, Takemoto C, Kojima S, Kawai G, Nyborg J, Watanabe K, Kaziro Y, Ohtsuki T

[J Biol Chem, 2001]

We have found the gene for a translation elongation factor Tu (EF-Tu) homologue in the genome of the nematode Caenorhabditis elegans. Because the corresponding protein was detected immunologically in a nematode mitochondrial (mt) extract, it could be regarded as a nematode mt EF-Tu. The protein possesses an extension of about 57 amino acids (we call this domain 3') at the C terminus, which is not found in any other known EF-Tu. Because most nematode mt tRNAs lack a T stem, domain 3' may be related to this feature. The nematode EF-Tu bound to nematode T stem-lacking tRNA, but bacterial EF-Tu was unable to do so. A series of domain exchange experiments strongly suggested that domains 3 and 3' are essential for binding to T stem-lacking tRNAs, This finding may constitute a novel example of the co-evolution of a structurally simplified RNA and the cognate RNA-binding protein, the latter having apparently acquired an additional domain to compensate for the lack of a binding site(s) on the RNA.

Ohtsuki T et al. (2007) IUBMB Life "T-armless tRNAs and elongated elongation factor Tu."

Ohtsuki T, Watanabe Y

[IUBMB Life, 2007]

Most tRNAs share a common secondary structure containing a T arm, a D arm, an anticodon arm and an acceptor stem. However, there are some exceptions. Most nematode mitochondrial tRNAs and some animal mitochondrial tRNAs lack the T arm, which is necessary for binding to canonical elongation factor Tu (EF-Tu). The mitochondria of the nematode Caenorhabditis elegans have a unique EF-Tu, named EF-Tu1, whose structure has supplied clues as to how truncated tRNAs can work in translation. EF-Tu1 has a C-terminal extension of about 60 aa that is absent in canonical EF-Tu. Recent data from our laboratory strongly suggests that EF-Tu1 recognizes the D-arm instead of the T arm by a mechanism involving this C-terminal region. Further biochemical analysis of mitochondrial tRNAs and EF-Tu from the distantly related nematode Trichinella spp. and sequence information on nuclear and mitochondrial DNA in arthropods suggest that T-armless tRNAs may have arisen as a result of duplication of the EF-Tu gene. These studies provide valuable insights into the co-evolution of RNA and RNA-binding proteins. IUBMB Life, 59: 68-75, 2007.

Sato A et al. (2006) Biochemistry "Identification of the residues involved in the unique serine specificity of Caenorhabditis elegans mitochondrial EF-Tu2."

Suzuki T, Watanabe K, Ohtsuki T, Sato A, Komiyama M, Watanabe YI

[Biochemistry, 2006]

In canonical translation systems, the single elongation factor Tu (EF-Tu) recognizes all elongator tRNAs. However, in Caenorhabditis elegans mitochondria, two distinct EF-Tu species, EF-Tu1 and EF-Tu2, recognize 20 species of T armless tRNA and two species of D armless tRNA(Ser), respectively. We previously reported that C. elegans mitochondrial EF-Tu2 specifically recognizes the serine moiety of serylated-tRNA. In this study, to identify the critical residues for the serine specificity in EF-Tu2, several residues in the amino acid binding pocket of bacterial EF-Tu were systematically replaced with corresponding EF-Tu2 residues, and the mutants were analyzed for their specificity for esterified amino acids attached to tRNAs. In this way, we obtained a bacterial EF-Tu mutant that acquired serine specificity after the introduction of 10 EF-Tu2 residues into its amino acid binding pocket. C. elegans EF-Tu2 mutants lacking serine specificity were also created by replacing seven or eight residues with bacterial residues. Further stressing the importance of these residues, we found that they are almost conserved in EF-Tu2 sequences of closely related nematodes. Thus, these three approaches reveal the critical residues essential for the unique serine specificity of C. elegans mitochondrial EF-Tu2.

Sakurai M et al. (2006) Biochem J "A protein extension to shorten RNA: elongated elongation factor-Tu recognizes the D-arm of T-armless tRNAs in nematode mitochondria."

Watanabe K, Watanabe YI, Sakurai M, Ohtsuki T

[Biochem J, 2006]

Nematode mitochondria possess extremely truncated tRNAs. Twenty of 22 tRNAs lack the entire T-arm. The T arm is necessary for the binding of canonical tRNAs and elongation factor (EF)-Tu. The nematode mitochondrial translation system employs two different EF-Tu factors named EF-Tu1 and EF-Tu2. Our previous study showed that nematode Caenorhabditis elegans EF-Tu1 binds specifically to T-armless tRNA. C. elegans EF-Tu1 has a 57-amino acid C-terminal extension that is absent from canonical EF-Tu, and the T-arm binding residues of canonical EF-Tu are not conserved. In this study, the recognition mechanism of T-armless tRNA by EF-Tu1 was investigated. Both modification interference assays and primer extension analysis of cross-linked ternary complexes revealed that EF-Tu1 interacts not only with the tRNA acceptor stem but also with the D arm. This is the first example of an EF-Tu recognizing the D-arm of a tRNA. The binding activity of EF-Tu1 was impaired by deletion of only 14 residues from the C-terminus, indicating that the C-terminus of EF-Tu1 is required for its binding to T-armless tRNA. These results suggest that C. elegans EF-Tu1 recognizes the D-arm instead of the T-arm by a mechanism involving its C-terminal region. This study sheds light on the co-evolution of RNA and RNA-binding proteins in nematode mitochondria.

Suematsu T et al. (2005) Nucleic Acids Res "A unique tRNA recognition mechanism of Caenorhabditis elegans mitochondrial EF-Tu2."

Ohtsuki T, Watanabe K, Sato A, Suematsu T, Sakurai M

[Nucleic Acids Res, 2005]

Nematode mitochondria expresses two types of extremely truncated tRNAs that are specifically recognized by two distinct elongation factor Tu (EF-Tu) species named EF-Tu1 and EF-Tu2. This is unlike the canonical EF-Tu molecule that participates in the standard protein biosynthesis systems, which basically recognizes all elongator tRNAs. EF-Tu2 specifically recognizes Ser-tRNA(Ser) that lacks a D arm but has a short T arm. Our previous study led us to speculate the lack of the D arm may be essential for the tRNA recognition of EF-Tu2. However, here, we showed that the EF-Tu2 can bind to D arm-bearing Ser-tRNAs, in which the D-T arm interaction was weakened by the mutations. The ethylnitrosourea-modification interference assay showed that EF-Tu2 is unique, in that it interacts with the phosphate groups on the T stem on the side that is opposite to where canonical EF-Tu binds. The hydrolysis protection assay using several EF-Tu2 mutants then strongly suggests that seven C-terminal amino acid residues of EF-Tu2 are essential for its aminoacyl-tRNA-binding activity. Our results indicate that the formation of the nematode mitochondrial (mt) EF-Tu2/GTP/aminoacyl-tRNA ternary complex is probably supported by a unique interaction between the C-terminal extension of EF-Tu2 and the tRNA.

Albertson DG et al. (1986) Worm Breeder's Gazette "progress in mapping genes."

Sulston JE, Fishpool RM, Albertson DG, Coulson AR

[Worm Breeder's Gazette, 1986]

Cloned genes are readily and routinely mapped to linkage groups of C. tu hybridization using the corresponding cosmids as probe DNAs. Our progress is summarized below. A few genetic markers (lower case letters above the line) have been included for reference. Uppercase letters above the line represent cloned DNAs corresponding to genes that have been sent to Cambridge. Genes designated by uppercase letters within error bars below the line are those mapped by in situ hybridization.