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BMC Res Notes. 2020 Jan 7;13(1):16. doi: 10.1186/s13104-019-4879-7.

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

Author information

1
School of Biological Sciences, Univ. Nebraska-Lincoln, Lincoln, NE, 68588-0118, USA. cchia1@unl.edu.
2
Department of Biological Sciences, Univ. Alabama in Huntsville, Huntsville, AL, 35899, USA.
3
iXpressGenes Inc, Hudson Alpha Institute for Biotechnology, 601 Genome Way, Huntsville, AL, 35806, USA.
4
School of Biological Sciences, Univ. Nebraska-Lincoln, Lincoln, NE, 68588-0118, USA.
5
Geisel School of Medicine, Dept. Molecular and Systems Biology, Dartmouth College, Hanover, NH, 03755, USA.

Abstract

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 fulfills 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 identifies a gene encoding a polypeptide containing the five 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.

KEYWORDS:

Dictyostelium discoideum; GFP; Mitochondrial targeting sequence; dUTPase

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