Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

Chia, Catherine P.; Inoguchi, Noriko; Varon, Kyle C.; Bartholomai, Bradley M.; Moriyama, Hideaki

doi:10.1186/s13104-019-4879-7

Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

Journal Article · Mon Jan 06 23:00:00 EST 2020 · BMC Research Notes

DOI:https://doi.org/10.1186/s13104-019-4879-7· OSTI ID:1629663

^[1]; Inoguchi, Noriko ^[2]; Varon, Kyle C. ^[3]; Bartholomai, Bradley M. ^[4]; Moriyama, Hideaki ^[3]

Univ. of Nebraska, Lincoln, NE (United States). School of Biological Sciences; DOE/OSTI
Univ. of Alabama, Huntsville, AL (United States). Dept. of Biological Sciences; iXpressGenes Inc, Hudson Alpha Inst. for Biotechnology, Huntsville, AL (United States)
Univ. of Nebraska, Lincoln, NE (United States). School of Biological Sciences
Dartmouth College, Hanover, NH (United States). Geisel School of Medicine. Dept. of Molecular and System Biology

Objective: The nuclear and mitochondrial genomes of Dictyostelium discoideum, a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectively. To begin to investigate how the pyrimidine biosynthetic pathway fulflls the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. Results: The annotated genome of D. discoideum identifes a gene encoding a polypeptide containing the fve conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the shortened N-termini that arose from the loss of residues 1-37. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1629663

Alternate ID(s):: OSTI ID: 1833655

Journal Information:: BMC Research Notes, Journal Name: BMC Research Notes Journal Issue: 1 Vol. 13; ISSN 1756-0500

Publisher:: BioMed CentralCopyright Statement

Country of Publication:: United States

Language:: English

References (39)

One Stop Shop for Everything Dictyostelium: dictyBase and the Dicty Stock Center in 2012 Fey, Petra; Dodson, Robert J.; Basu, Siddhartha Methods in Molecular Biology https://doi.org/10.1007/978-1-62703-302-2_4	book	January 2013
Processing of mitochondrial presequences Mossmann, Dirk; Meisinger, Chris; Vögtle, F. -Nora Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Vol. 1819, Issue 9-10 https://doi.org/10.1016/j.bbagrm.2011.11.007	journal	September 2012
Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum. Troll, H.; Malchow, D.; Müller-Taubenberger, A. Journal of Biological Chemistry, Vol. 267, Issue 29 https://doi.org/10.1016/s0021-9258(19)36799-7	journal	October 1992
Human dUTP pyrophosphatase: uracil recognition by a β hairpin and active sites formed by three separate subunits Mol, Clifford D.; Harris, Jonathan M.; McIntosh, Evan M. Structure, Vol. 4, Issue 9 https://doi.org/10.1016/s0969-2126(96)00114-1	journal	September 1996
Global analysis of protein expression in yeast Ghaemmaghami, Sina; Huh, Won-Ki; Bower, Kiowa Nature, Vol. 425, Issue 6959 https://doi.org/10.1038/nature02046	journal	October 2003
Structure of ATP citrate lyase and the origin of citrate synthase in the Krebs cycle Verschueren, Koen H. G.; Blanchet, Clement; Felix, Jan Nature, Vol. 568, Issue 7753 https://doi.org/10.1038/s41586-019-1095-5	journal	April 2019
Full-length transcriptome analysis of multiple organs and identification of adaptive genes and pathways in Mikania micrantha Ruan, Xiaoxian; Wang, Zhen; Su, Yingjuan Scientific Reports, Vol. 12, Issue 1 https://doi.org/10.1038/s41598-022-07198-0	journal	February 2022
Characterization of Distinct Nuclear and Mitochondrial Forms of Human Deoxyuridine Triphosphate Nucleotidohydrolase Ladner, Robert D.; McNulty, Dean E.; Carr, Steven A. Journal of Biological Chemistry, Vol. 271, Issue 13 https://doi.org/10.1074/jbc.271.13.7745	journal	March 1996
Identification of a Consensus Cyclin-dependent Kinase Phosphorylation Site Unique to the Nuclear Form of Human Deoxyuridine Triphosphate Nucleotidohydrolase Ladner, Robert D.; Carr, Steven A.; Huddleston, Michael J. Journal of Biological Chemistry, Vol. 271, Issue 13 https://doi.org/10.1074/jbc.271.13.7752	journal	March 1996
Kinetic Characterization of dUTPase from Escherichia coli Larsson, Gunilla; Nyman, Per Olof; Kvassman, Jan-Olov Journal of Biological Chemistry, Vol. 271, Issue 39 https://doi.org/10.1074/jbc.271.39.24010	journal	September 1996
Developmental Regulation of dUTPase in Drosophila melanogaster Békési, Angéla; Zagyva, Imre; Hunyadi-Gulyás, Éva Journal of Biological Chemistry, Vol. 279, Issue 21 https://doi.org/10.1074/jbc.m313647200	journal	May 2004
The prediction of organelle-targeting peptides in eukaryotic proteins with Grammatical-Restrained Hidden Conditional Random Fields Indio, Valentina; Martelli, Pier Luigi; Savojardo, Castrense Bioinformatics, Vol. 29, Issue 8 https://doi.org/10.1093/bioinformatics/btt089	journal	February 2013
Protein sequence comparisons show that the ‘pseudoproteases’ encoded by poxviruses and certain retroviruses belong to the deoxyuridine triphosphatase family McGeoch, Duncan J. Nucleic Acids Research, Vol. 18, Issue 14 https://doi.org/10.1093/nar/18.14.4105	journal	January 1990
Dictyostelium acetoacetyl-CoA thiolase is a dual-localizing enzyme that localizes to peroxisomes, mitochondria and the cytosol Sekiba, Atsushi; Nagayama, Koki; Itagaki, Shoko Microbiology, Vol. 161, Issue 7 https://doi.org/10.1099/mic.0.000102	journal	July 2015
Fumarase: a paradigm of dual targeting and dual localized functions: Fumarase: dual targeting and dual localized functions Yogev, Ohad; Naamati, Adi; Pines, Ophry FEBS Journal, Vol. 278, Issue 22 https://doi.org/10.1111/j.1742-4658.2011.08359.x	journal	October 2011
Structural insights into the mechanism defining substrate affinity in Arabidopsis thaliana dUTPase: the role of tryptophan 93 in ligand orientation Inoguchi, Noriko; Chaiseeda, Kittichai; Yamanishi, Mamoru BMC Research Notes, Vol. 8, Issue 1 https://doi.org/10.1186/s13104-015-1760-1	journal	December 2015
Tissue-specific G1-phase cell-cycle arrest prior to terminal differentiation in Dictyostelium Chen, G. Development, Vol. 131, Issue 11 https://doi.org/10.1242/dev.01151	journal	June 2004
Live imaging of the Dictyostelium cell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint Muramoto, T.; Chubb, J. R. Development, Vol. 135, Issue 9 https://doi.org/10.1242/dev.020115	journal	March 2008
Phosphorylated peptide of G protein-coupled receptor induces dimerization in activated arrestin Stadler, Andreas M.; Granzin, Joachim; Cousin, Anneliese RWTH Aachen University https://doi.org/10.18154/rwth-2020-08655	text	January 2020
Homotrimeric dUTPases; Structural Solutions for Specific Recognition and Hydrolysis of dUTP Persson, R.; Cedergren-zeppezauer, E.; Wilson, K. Current Protein & Peptide Science, Vol. 2, Issue 4 https://doi.org/10.2174/1389203013381035	journal	December 2001
Flexible glycine rich motif ofEscherichia coli deoxyuridine triphosphate nucleotidohydrolase is important for functional but not for structural integrity of the enzyme Vertessy, Beata G. Proteins: Structure, Function, and Genetics, Vol. 28, Issue 4 https://doi.org/10.1002/(SICI)1097-0134(199708)28:4<568::AID-PROT10>3.0.CO;2-E	journal	August 1997
Dictionary of protein secondary structure: Pattern recognition of hydrogen-bonded and geometrical features Kabsch, Wolfgang; Sander, Christian Biopolymers, Vol. 22, Issue 12 https://doi.org/10.1002/bip.360221211	journal	December 1983
Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum. Troll, H.; Malchow, D.; Müller-Taubenberger, A. Journal of Biological Chemistry, Vol. 267, Issue 29 https://doi.org/10.1016/S0021-9258(19)36799-7	journal	October 1992
Transmembrane domains in the functions of Fc receptors Zidovetzki, Raphael; Rost, Burkhard; Armstrong, Don L. Biophysical Chemistry, Vol. 100, Issue 1-3 https://doi.org/10.1016/S0301-4622(02)00306-X	journal	December 2002
Human dUTP pyrophosphatase: uracil recognition by a β hairpin and active sites formed by three separate subunits Mol, Clifford D.; Harris, Jonathan M.; McIntosh, Evan M. Structure, Vol. 4, Issue 9 https://doi.org/10.1016/S0969-2126(96)00114-1	journal	September 1996
Keeping Uracil Out of DNA: Physiological Role, Structure and Catalytic Mechanism of dUTPases Vértessy, Beáta G.; Tóth, Judit Accounts of Chemical Research, Vol. 42, Issue 1 https://doi.org/10.1021/ar800114w	journal	January 2009
Global analysis of protein localization in budding yeast Huh, Won-Ki; Falvo, James V.; Gerke, Luke C. Nature, Vol. 425, Issue 6959 https://doi.org/10.1038/nature02026	journal	October 2003
The genome of the social amoeba Dictyostelium discoideum Eichinger, L.; Pachebat, J. A.; Glöckner, G. Nature, Vol. 435, Issue 7038 https://doi.org/10.1038/nature03481	journal	May 2005
BaCelLo: a Balanced subCellular Localization predictor. Pierleoni, Andrea; Martelli, Pier Luigi; Fariselli, Piero Protocol Exchange https://doi.org/10.1038/nprot.2007.165	journal	March 2007
Systematic discovery of pseudomonad genetic factors involved in sensitivity to tailocins Carim, Sean; Azadeh, Ashley L.; Kazakov, Alexey E. The ISME Journal, Vol. 15, Issue 8 https://doi.org/10.1038/s41396-021-00921-1	journal	March 2021
Mitochondrial DNA replication but no nuclear DNA replication during development of Dictyostelium. Shaulsky, G.; Loomis, W. F. Proceedings of the National Academy of Sciences, Vol. 92, Issue 12 https://doi.org/10.1073/pnas.92.12.5660	journal	June 1995
The Human dUTPase Gene Encodes both Nuclear and Mitochondrial Isoforms Ladner, Robert D.; Caradonna, Salvatore J. Journal of Biological Chemistry, Vol. 272, Issue 30 https://doi.org/10.1074/jbc.272.30.19072	journal	July 1997
Developmental Regulation of dUTPase in Drosophila melanogaster Békési, Angéla; Zagyva, Imre; Hunyadi-Gulyás, Éva Journal of Biological Chemistry, Vol. 279, Issue 21 https://doi.org/10.1074/jbc.M313647200	journal	May 2004
MitoFates: Improved Prediction of Mitochondrial Targeting Sequences and Their Cleavage Sites Fukasawa, Yoshinori; Tsuji, Junko; Fu, Szu-Chin Molecular & Cellular Proteomics, Vol. 14, Issue 4 https://doi.org/10.1074/mcp.M114.043083	journal	February 2015
BaCelLo: a balanced subcellular localization predictor Pierleoni, A.; Martelli, P. L.; Fariselli, P. Bioinformatics, Vol. 22, Issue 14 https://doi.org/10.1093/bioinformatics/btl222	journal	July 2006
MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability Katoh, K.; Standley, D. M. Molecular Biology and Evolution, Vol. 30, Issue 4 https://doi.org/10.1093/molbev/mst010	journal	January 2013
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Altschul, Stephen F.; Madden, Thomas L.; Schäffer, Alejandro A. Nucleic Acids Research, Vol. 25, Issue 17, p. 3389-3402 https://doi.org/10.1093/nar/25.17.3389	journal	September 1997
A series of PDB-related databanks for everyday needs Touw, Wouter G.; Baakman, Coos; Black, Jon Nucleic Acids Research, Vol. 43, Issue D1 https://doi.org/10.1093/nar/gku1028	journal	October 2014
Computational Method to Predict Mitochondrially Imported Proteins and their Targeting Sequences Claros, Manuel G.; Vincens, Pierre European Journal of Biochemistry, Vol. 241, Issue 3 https://doi.org/10.1111/j.1432-1033.1996.00779.x	journal	November 1996

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Dictyostelium discoideum
GFP
Mitochondrial targeting sequence
dUTPase

MOESM1 of Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus Chia, Catherine; Inoguchi, Noriko; Varon, Kyle https://doi.org/10.6084/m9.figshare.11543322 The current record is supplemented by this text	text	January 2020
MOESM1 of Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus Chia, Catherine; Inoguchi, Noriko; Varon, Kyle https://doi.org/10.6084/m9.figshare.11543322.v1 The current record is supplemented by this text	text	January 2020
MOESM2 of Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus Chia, Catherine; Inoguchi, Noriko; Varon, Kyle https://doi.org/10.6084/m9.figshare.11543331 The current record is supplemented by this text	text	January 2020
MOESM2 of Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus Chia, Catherine; Inoguchi, Noriko; Varon, Kyle https://doi.org/10.6084/m9.figshare.11543331.v1 The current record is supplemented by this text	text	January 2020
MOESM3 of Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus Chia, Catherine; Inoguchi, Noriko; Varon, Kyle https://doi.org/10.6084/m9.figshare.11543340 The current record is supplemented by this text	text	January 2020
MOESM3 of Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus Chia, Catherine; Inoguchi, Noriko; Varon, Kyle https://doi.org/10.6084/m9.figshare.11543340.v1 The current record is supplemented by this text	text	January 2020
Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus Chia, Catherine; Inoguchi, Noriko; Varon, Kyle https://doi.org/10.6084/m9.figshare.c.4808679 The current record is supplemented by this collection	collection	January 2020
Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus Chia, Catherine; Inoguchi, Noriko; Varon, Kyle https://doi.org/10.6084/m9.figshare.c.4808679.v1 The current record is supplemented by this collection	collection	January 2020

Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

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