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

doi:10.21203/rs.2.13015/v1

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

https://doi.org/10.21203/rs.2.13015/v1

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Objective The nuclear and mitochondrial genomes of Dictyostelium discoideum , a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectivey. 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 truncated N-termini. 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.

Molecular Biology

dUTPase

Dictyostelium discoideum

GFP

mitochondrial targeting sequence

The nuclear and mitochondrial genomes of Dictyostelium discoideum are 78% and 73% AT, respectively [1, 2], creating a substantial requirement for dUMP, the precursor for dTTP, during mitotic cell growth as well as during development when DNA replication also occurs [3–5]. To understand how the pyrimidine biosynthesis pathway accommodates the demand for dTTP, we began to focus on a key enzyme of the pathway, deoxyuridine triphosphate nucleotidohydrolase or dUTPase, which hydrolyzes dUTP to pyrophosphate and dUMP; dUMP is subsequently converted to dTTP. Concomitantly, a high dTTP to dUTP ratio is ensured, thus minimizing the incorporation of uracil during DNA synthesis [6].

The curated genome of the soil amoeba D. discoideum shows a single gene (DictyBase Gene ID DDB_G0293374; [7]) predicted to encode a dUTPase polypeptide containing the five hallmark motifs (M1-M5) of homotrimeric dUTPases [8], seen in the alignments of the amino acid sequences from mustard, yeast and human (Fig. 1a). While the dUTPases of Arabidopsis thaliana and D. discoideum have substantial stretches of identity (73%) within the 138-residue segment containing M1-M5 [9], their N-termini have very low sequence similarity to each other, and to the human and yeast N-termini. Notably, within the lengthy N-terminus of the D. discoideum dUTPase, atypical of most dUTPases, computational analyses predict a mitochondrial targeting sequence (MTS).

Results

A single gene codes for an active enzyme in the homotrimeric dUTPase family

To characterize the unusual D. discoideum dUTPase, we established first that the recombinant protein was an active dUTPase and determined its kinetic parameters. Also catalytically-active was a core version comprised of polypeptide subunits that retain M1-M5 but lack residues 1–37 of the N-terminus. A homotrimeric structure of the core dUTPase enzyme was confirmed by crystallographic analyses that additionally showed interactions of the truncated N-termini with the C-termini likely enhanced substrate access of the dUTP substrate to the active sites.

Recombinant His-tagged dUTPase proteins were expressed in E. coli, purified by metal-chelating chromatography and after removal of the His-tag, assayed for activity by measuring the release of protons with the pH indicator cresol red (SM Text S1). As determined by the kinetic parameters calculated from stopped-flow measurements (Fig. 1b, c and Table 1), the k_cat/K_M of the core protein, lacking N-terminal residues 1–37 (Fig. 1a), was 60-fold greater than that of the full-length species, indicating the core was a more efficient enzyme.

Table 1 Calculated kinetic parameters of D. discoideum dUTPases

K_M

V_max

mM·s^-¹

k_cat*

s^-1

k_cat/K_M

µM^-1s^-1

core

0.5 ± 0.1

1.4 ± 0.06

9.3

18.6

full-length

1.0 ± 0.2

0.5 ± 0.03

3.3

0.3

*k_cat= V_max/[E_T]; [E_T]=0.15 mM

Activity assays using an end-point method showed the full-length and core dUTPases both were metal-dependent enzymes inhibited by EDTA. Mg²⁺was identified as the optimal divalent cation (SM Table S1), as seen with homotrimeric dUTPases [6]. Both proteins specifically used dUTP (≤1% hydrolysis of other dNTPs; data not shown) and exhibited optimal activities at 60˚C and pH 8 (SM Fig. S1). The catalytically efficient core dUTPase indicated that the 37 N-terminal residues were not essential for activity.

The crystal structure of the core dUTPase showed it to be a homotrimer, indicating that the absent 37 N-terminal residues were dispensable for oligomer formation (SM Fig. S2; PDB ID 5F9K). In most homotrimeric dUTPases, the C-termini, containing M5 which aids in coordinating the ligand at the active site, are flexible. In the core structure, PISA interaction analyses revealed that the shorter N-termini interacted with the C-termini of adjacent subunits, constraining their engagement with their respective active sites. The interactions are schematically shown for Chains A and C, and Chains B and A (Fig. 1d). The limited movement of the C-termini contributed to the different inhibitor coordinates in each active site of the core dUTPase (SM Fig. S2, insets). Notable is the position of Inhibitor B. Interactions (hydrogen bonds and salt bridges) by three glutamates of the C-terminus of Chain C with residues of the N-terminus of Chain A (E120::H3+F5, E123::K5 and E126::K9) produced the most displaced orientation compared to Inhibitors A and C (detailed in SM Fig. 3). The C-termini, which act normally as lids for the substrate binding pockets [10–12], instead were restricted in their movement, which may have allowed increased access of dUTP to the three active sites compared to the full-length protein with its extended N-termini. We postulate this to be why the core enzyme was more catalytically efficient in vitro compared to the full-length dUTPase.

The N-terminus of D. discoideum dUTPase contained a mitochondrial targeting sequence

The role of the N-terminus of the D. discoideum dUTPase was explored first by expressing in Ax2 cells a full-length dUTPase with GFP at the C-terminus of the polypeptide subunit (Fig. 2a). By microscopy, the fusion protein was observed to localize to Mitotracker™–labeled mitochondria (Fig. 2b), supporting the hypothesis that N-terminus contained an MTS. Contrary to expectations, nuclei did not display any GFP fluorescence (Fig. 2c). A helical wheel projection showed that N-terminal residues 1–20 of dUTPase formed an amphipathic helix [13] (Fig. 2e), with a net positive charge, a characteristic common to presequences of imported mitochondrial proteins [14]. Programs predicting targeting sequences indicated a high probability of dUTPase to be located in mitochondria, when fungi were used in the organism category, with cleavage after Leu18 upon import into the organelle (BaCelLo [15], MitoFates [16], MitoProt II [17]).

As a test for the presence of an MTS within the N-terminus of dUTPase, we added the forty N-terminal residues of dUTPase to GFP, producing N_1–40-dUTPase-GFP (Fig. 2a). Like the full-length dUTPase-GFP fusion protein, the expressed N_1–40-dUTPase-GFP co-localized with Mitotracker™–labeled mitochondria (Fig. 2d), reinforcing the prediction of an MTS within the N-terminus of dUTPase. The majority of the mitochondria had both GFP and Mitotracker™ signals, but not all Mitotracker™–stained mitochondria had a GFP signal. No fluorescence was observed in nuclei (data not shown).

Protein blots, of whole cell lysates and mitochondria prepared from cells expressing N_1–40-dUTPase-GFP, were probed with antibodies against porin, a mitochondria-specific protein [18] or GFP (Fig. 2f). As expected, the signal for anti-porin was enhanced in the mitochondria preparation (upper panel). Observed also in mitochondria was a strong anti-GFP signal (lower panel) that corroborated the microscopy images, indicating that N_1–40-dUTPase-GFP localized to mitochondria. The two anti-GFP signals observed both in the lysate and mitochondria were interpreted to be the entire N_1–40-dUTPase-GFP, predicted to be 32.5 kDa and a processed version of it, migrating at 27.5 kDa. The approximate size difference of 5 kDa corresponded to the predicted 4.8 kDa mass of the forty residues of dUTPase fused to GFP, suggesting the loss of the dUTPase amino acids after import into the organelle.

To determine that cleavage of N_1–40-dUTPase-GFP occurred, we obtained the N-terminal sequence of the 27.5 kDa protein immunoprecipitated from cell lysates using anti-GFP antibodies. The processed fusion protein was cleaved after Gly41, the first glycine of the (Gly-Ala)₅ linker between the dUTPase and GFP sequences (Fig. 2a), confirming the loss of all forty dUTPase residues. Though within the range of presequence lengths, this was lengthier than the predicted presequence that identified cleavage after Leu18, a residue common within an R–2 motif recognized by the mitochondrial processing peptidase [16, 19] [reviewed in 14]. An intermediate was not detected by immunoblotting, but it is possible that the fusion protein was cleaved twice, once after Leu18 and then after Gly41, generating the final A(GA)₄-GFP molecule inside the mitochondria.

Summary

Activity and structural analyses of the D. discoideum dUTPase showed it to be a functional enzyme with attributes typical of most eukaryotic dUTPases. The unusually long N-terminus of the polypeptide subunit was found to be dispensable for catalytic activity as well as homotrimer formation. Microscopy and biochemical evidence showed that fusion proteins, where GFP was attached to the full-length polypeptide or the forty N-terminal amino acids of dUTPase, localized to mitochondria, but not to nuclei, indicating the presence of an MTS within the N-terminus of the D. discoideum dUTPase.

The presence of dUTPase in D. discoideum mitochondria is reasonable given the needed synthesis of nucleotides for replication of the organellar DNA that is 73% AT. Based on its nuclear presence in yeast, flies and humans [20–23], we also expected to see dUTPase in nuclei, but did not observe a GFP signal in nuclei of D. discoideum cells of log-phase cultures expressing the full-length dUTPase-GFP fusion protein (Fig. 2c). Technical reasons could account for an absence of a nuclear signal. Fused to the C-terminus and larger than an individual dUTPase subunit (mass of the predicted full-length polypeptide is 19.8 kDa), the GFP sequence (27.6 kDa) may have interfered with homotrimer assembly or masked nuclear localization signals. The successful cytological detection of dUTPase in the nucleus may require a construct with a small tag that is less likely to disrupt folding or cover signal epitopes on the dUTPase surface, coupled with methods that synchronize cell division or identify cells entering S-phase when presumably the nucleotide biosynthesis enzymes are actively providing dNTPs for DNA replication.

Alternatively, a nuclear-localized dUTPase in D. discoideum may be a location-specific isoform, rather than the predicted full-length protein, as seen for the nuclear dUTPase proteins of H. sapiens and D. melanogaster [20, 24]. Examples in D. discoideum where two versions of a protein are generated from single copy genes include (peroxisomal and mitochondrial) isoforms of the enzyme acetoacetyl-CoA thiolase (DDB_G0271544) that arise from alternative start codons [25] and (cytoplasmic and mitochondrial) isoforms of fumarase (DDB_G0280495), inferred from expressed sequence tag data. Fumarase isoforms have been documented extensively in yeast and mammals [reviewed in 26].

An increasing number of eukaryotic proteins are reported to have more than one subcellular location, and enzymes of nucleotide synthesis pathways are candidates for multiple locations since DNA replication occurs in the nucleus, mitochondria and chloroplasts. The mitochondrial location of the D. discoideum dUTPase, and the previously identified mitochondrial and nuclear isoforms of the human dUTPase, raises questions of whether other enzymes in the pyrimidine nucleotide biosynthesis pathway are similarly positioned and whether other eukaryotes share this pattern of multiple locations. The genetically-tractable D. discoideum is an experimental system that can be exploited to determine and explore molecular mechanisms of the subcellular distribution of thymidylate synthase, dCMP and dCTP deaminases (supported by expressed sequence tag data), and should reveal whether these enzymes use targeting strategies that coordinate their distribution with that of dUTPase, ensuring dNTPs for the replication of nuclear and organellar DNA.

dUTPase deoxyuridine triphosphate nucleotidohydrolase

GFP green fluorescent protein

MTS mitochondrial targeting sequence

Ethics Approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and material

The structure of the D. discoideum core dUTPase was deposited in the Protein Data Bank with the ID 5F9K. The dataset supporting the conclusions of this article are included within the article and Supplementary Materials.

Competing Interests

The author(s) declare that they have no competing interests.

Funding

The John C. and Nettie V. David Memorial Trust Fund through a UNL Research Council Grant-in-Aid to CPC provided partial support of this work. B. Bartholomai received support from the UCARE Program at UNL and a Grant in Aid of Research from Sigma Xi.

Authors’ contributions

CPC wrote the manuscript with major contributions to the crystallography analyses from NI and HM. NI and HM performed crystallography and analyses of the 3D structure of the core dUTPase. BB performed microscopy imaging and assisted CPC with DNA cloning. CPC and KCV purified proteins. CPC, HM and KV measured and calculated enzyme activities. All authors read and approved the final manuscript.

Acknowledgements

This research used resources from the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02–06CH11357. We thank: Dr. Javier Seravalli (Dept. of Biochemistry, UNL) for his contributions to the stopped-flow activity assays; Dr. Jay Dunlap, Dr. Jennifer Loros, and the Microscopy Core (Dartmouth) for allowing BMB to carry out follow-up microscopy imaging for this project while pursuing his PhD thesis work.

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

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