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 · Tue Jan 07 04:00:00 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

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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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