Nuclear localization signal-dependent and -independent movements of Drosophila melanogaster dUTPase isoforms during nuclear cleavage

Biochemical and Biophysical Research Communications 381 (2009) 271–275

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Nuclear localization signal-dependent and -independent movements of Drosophila melanogaster dUTPase isoforms during nuclear cleavage } Muha a, Imre Zagyva a, Zsolt Venkei b, János Szabad b, Beáta G. Vértessy a,* Villo a b

Institute of Enzymology, Hungarian Academy of Sciences, Laboratory of Genome Metabolism and Repair, Karolina út 29, H-1113, P.O. Box 7, H-1518 Budapest, Hungary Department of Biology, Faculty of Medicine, University of Szeged, Szeged, Hungary

a r t i c l e

i n f o

Article history: Received 5 February 2009 Available online 14 February 2009

Keywords: Nuclear cleavage Nucleo-cytoplasmic transport dUTPase Nuclear localization signal

a b s t r a c t Two dUTPase isoforms (23 kDa and 21 kDa) are present in the fruitfly with the sole difference of an Nterminal extension. In Drosophila embryo, both isoforms are detected inside the nucleus. Here, we investigated the function of the N-terminal segment using eYFP–dUTPase constructs. In Schneider 2 cells, only the 23 kDa construct showed nuclear localization arguing that it may contain a nuclear localization signal (NLS). Sequence comparisons identified a lysine-rich nonapeptide with similarity to the human c-myc NLS. In Drosophila embryos during nuclear cleavages, the 23 kDa isoform showed the expected localization shifts. Contrariwise, although the 21 kDa isoform was excluded from the nuclei during interphase, it was shifted to the nucleus during prophase and forthcoming mitotic steps. The observed dynamic localization character showed strict timing to the nuclear cleavage phases and explained how both isoforms can be present within the nuclear microenvironment, although at different stages of cell cycle. Ó 2009 Elsevier Inc. All rights reserved.

Introduction The uracil-containing pyrimidine mononucleotide dUMP is not a normal constituent of DNA, although uracil is equivalent to thymine in Watson–Crick base-pairing [1]. This negative discrimination is probably due to the high frequency of cytosine deamination events that result in mispaired uracil [2]. In almost all organisms, uracil-substituted DNA is subjected to base-excision repair initiated by uracil-DNA glycosylases [3,4]. An efficient preventive repair pathway is also generally present that keeps cellular dUTP/dTTP levels at a much decreased value to prevent use of dUTP as a building block by DNA polymerases. The enzyme responsible for this sanitizing role is dUTPase, a ubiquitous protein encoded in bacteria and eukaryotes alike and even in distinct families of retroviruses [5,6]. Lack of dUTPase results in high uracil-content within DNA that triggers transformation of base-excision repair into a hyperactive futile cycle leading to chromosome fragmentation and thymine-less cell death [7]. All eukaryotic dUTPases, except from several parasites [8], are homotrimers with three active sites built from conserved sequence motifs of each subunits [6,9–12]. In some eukaryotes, two dUTPase isoenzymes are generated by mRNA alternative splicing [13] or by use of alternative promoters

Abbreviations: dUTPase, dUTP pyrophosphatase (EC 3.6.1.23); NLS, nuclear localization signal; NPC, nuclear pore complex. * Corresponding author. Fax: +36 1 466 5465. E-mail address: [email protected] (B.G. Vértessy). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.02.036

[14]. In the latter case, human cells contain nuclear and mitochondrial isoforms possessing cognate localization signals. The nuclear isoform is under cell-cycle control while the mitochondrial isoform is constitutive [14], probably reflecting the semi-independent nature of mitochondrial DNA repair. The experimentally verified nuclear localization signal observed in nuclear isoform of human dUTPase [15] is rather unusual but shows similarity to c-myc and and RanBP nuclear localization signals [16,17]. Drosophila melanogaster cells also contain two dUTPase isoforms [13] (23 kDa and 21 kDa, which will correspond to two physiological trimeric enzymes of 69 kDa and 63 kDa, respectively). These are generated by alternative splicing but are both expressed under the control of the same cell-cycle-dependent promoter and both are therefore expected to be present only in actively dividing cells [13]. The 23 kDa isoform contains an N-terminal extension of 13 amino acid residues. In localization studies carried out with ovaries, embryos and larvae; immunohistochemistry and Western blotting of nuclear, cytoplasmic and mitochondrial extracts indicated that both Drosophila dUTPase isoforms can be present either in the nucleus or in the cytoplasm in different tissues and developmental stages [13] preventing clear assignment of the two isoforms to distinct dedicated cellular subcompartments. Nuclear trafficking of proteins larger than approximately 40 kDa is carried out by a dedicated transport mechanism. It involves importin proteins in GDP/GTP complexes that cycle through the nuclear pore complex (NPC) [18,19]. During mitosis, nuclear pore complexes disassemble and reassemble in each cycle at specific mitotic steps. Drosophila embryos offer a particularly useful

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model to study mitosis in the syncytial blastoderm stage where mitotic events are strongly synchronized within the embryo and the mitotic wave can be observed by confocal microscopy [20,21]. During nuclear cleavage in Drosophila embryos, the nuclear pore complexes disassemble but the nuclear envelope is partially retained and provides some separation between nuclear and cytoplasmic compartments. This specific pattern has been termed as ‘‘semi-closed” (the term ‘‘semi-open” is also used) mitosis [22] and the retained nuclear membrane is called spindle envelope. Macromolecules can pass through the spindle envelope in a practically unrestricted manner and the directional importin-dependent import mechanism does not operate. The goal of the present study was to use both S2 cells and embryos to describe cellular trafficking of Drosophila dUTPase isoforms that showed unexpected localization patterns in earlier studies [13]. Yellow-fluorescent protein (eYFP)-tagged 21 and 23 kDa isoform dUTPase constructs were overexpressed in S2 cells and their localization was followed in these cells as well as in microinjected embryos. Results indicate that trafficking of the 23 kDa isoform can be explained by the conventional model based on NLS-dependent movements through the nuclear pore complex. Within the N-terminal extension of the 23 kDa isoform, a lysinerich segment showed 66.7% identity to the NLS segment described in human c-myc [16]. For the 21 kDa isoform, however, NLS- and NPC-independent processes can be visualized. Results allow identification of an NLS segment conserved among dUTPases, and provide an experimental explanation to the previously observed unexpected localization patterns. Materials and methods Construction of 21 kDa and 23 kDa dUTPase reporter plasmids. pRmNDUT–eYFP (21 kDa dUTPase) and pRmDUT–eYFP (23 kDa dUTPase) vectors were constructed by cloning 21 kDa and 23 kDa dUTPase coding sequences into the Drosophila transfection vector pRm–eYFP–N–C* [23] The LDdut-pET22b [12] plasmid

was used as a template for amplification of D. mel. dUTPase gene by PCR. The PCR was performed with two different forward primers 50 -ctagctagcatgccatcaaccgatttcgccgacattc-30 (23 kDa) and 50 -ctagctagcatgaagatcgacacgtgcgtcctgcg-30 (21 kDa), and a single reverse primer 50 -atagtttagcggccgcgtagcaacaggagccggagc-30 . These primers introduce a NheI site upstream and a NotI site downstream of the gene. The pRmDUT–eYFP and pRmNDUT– eYFP constructs produce dUTPase with a C-terminal eYFP (enhanced yellow-fluorescent protein) under the control of a metallothionein promoter. S2 cell culturing, transfection, selection. Drosophila melanogaster Schneider 2 cells were grown as semi-adherent layers at 25 °C in serum free medium (Gibco) supplemented with 20 mM L-glutamine, 10 U/mL penicillin, 0.1 mg/mL streptomycin (Sigma). For stable transfection of S2 cells, dUTPase–eYFP expression constructs were cotransfected with pPURO plasmid in 1:20 molar ratio in the presence of Cellfectine (Invitrogen) following the manufacturer’s instructions. Stable cell lines were selected by growing them in the presence of increased concentration of puromycin. The final puromycin concentration was 50 lg/mL. Localization of dUTPase–YFP in S2 cells. For investigating the subcellular distribution of dUTPase isoforms, dUTPase–eYFP, DNA and actin were visualized. Selected S2 cells were cultured on microscope slides. Expression of dUTPase–eYFP constructs was induced at 25 °C by addition of 700 lM CuSO4 and over night incubation. Cells were washed with PBS, fixed in 3% paraformaldehyde for 5 min, and permeabilized with 0.1% Triton X-100. DNA was stained with DAPI (Sigma) and actin was labeled with rhodamine–phalloidine. Samples mounted in FluorSave Reagent (Calbiochem) were visualized with Olympus IX70 confocal laser scanning microscope, under a 60 oil immersion objective. Microinjection of S2 cell extract into Drosophila embryo, confocal microscopy. After ON induction of dUTPase–eYFP expression, S2 cells were washed off by pipetting and washed in PBS. Cell pellet was homogenized on ice in equal volume of lysis buffer (150 mM NaCl, 10% glicerin, 10 mM Tris, pH 7.4, 1 mM DTT, protease inhib-

23 kDa N’

PAAKKMKID

dUTPase conserved core

21 kDa specific segment

C’

N’

dUTPase conserved core

specific segment

C’

MKID

Fig. 1. Localization of the D. mel. dUTPase isoforms in S2 cells. (A) 23 kDa isoform. (B) 21 kDa isoform. The schematic representation of the 23 kDa and 21 kDa isoforms indicates that the 23 kDa isoform contains an additional N-terminal segment with a putative nuclear localization signal. Both isoforms contain a 28-residue-segment at the Cterminus that is found only in the Drosophila dUTPases. Note that the 23 kDa isoform shows nuclear localization while the 21 kDa isoform is cytoplasmic.

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itor), than lysate was cleared by centrifugation (14,300 rpm, 15 min, 4 °C). Rhodamine–tubulin was added to the S2 cell extract to a final concentration of in order to follow dynamics of microtubule assembly related to nuclear divisions. Approximately 200 pL of freshly prepared cell extract supplemented with rhodamine–tubulin (2% of total egg volume) was injected into the posterior region of wild-type (Oregon R) embryos at syncytial blastoderm stage (cycles 10–13). Embryos were dechorionated in 3% sodium hypochlorite before injection. Localization of the two dUTPase isoforms were followed separately over time by series of optical sections generated with an

Prophase

Metaphase

Results Localization of Drosophila dUTPase isoforms in S2 cells and identification of a putative NLS segment conserved among dUTPases Fig. 1 presents immunofluorescent micrographs of S2 cells overexpressing fluorescently labeled constructs of the 23 (A) and 21 kDa (B) dUTPase isoforms (green).1 The samples were also stained for DNA (blue) and actin (red), to aid interpretation of

Anaphase

Telophase

Cytokinesis

megre

tubulin

23kDa dUTPase

Interphase

Olympus VS1000 confocal microscope. The injections were carried out at 20 °C.

25µm

Prophase

Metaphase

Anaphase

Telophase

Cytokinesis

merge

tubulin

21kDa dUTPase

Interphase

25µm Fig. 2. Localization shifts of the two isoforms during the cell division cycle from interphase to cytokinesis. (A) 23 kDa isoform. (B) 21 kDa isoform. Note the opposing shifts of the two isoforms, evident from prophase.

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the results. Continuous selection after transfection resulted in approximately 70% of the cells overexpressing the fluorescent construct. The 21 kDa isoform is present around the nucleus and within the cytoplasm in all cells; it is, however, strictly excluded from the nuclei. This observation directly suggests that the N-terminal extension may contain a NLS segment, lack of which prevents nuclear import of dUTPase in all cell types present in the S2 cell lines. This suggestion is further supported by the experimental observation that the 23 kDa isoform with the N-terminal extension is visualized within the karyoplasm (Fig. 2A). Within the N-terminal extension of the 23 kDa Drosophila dUTPase sequence, an NLS segment was identified which shows very high similarity to the NLS of human c-myc oncoprotein [16] (Table 1). Both of these NLSs represent members of a rather unusual type Table 1 dUTPases possess an unusual NLS. (A) D. melanogaster dUTPase H. sapiens c-myc H. sapiens dUTPase H. sapiens RanBP3

PAAKKMKID PAAKRVKLD SPSKRARPA PPVKRERTS

(B) D. melanogaster dUTPase H. sapiens dUTPase M. musculus dUTPase R. norvegicus dUTPase G. gallus dUTPase C. elegans dUTPase L. esculentum dUTPase O. sativa dUTPase Consensus

PAAKKMKID SPSKRARPA SASKRARAE SVSKRARAE SPSKRQKGS PALKKSKTE PSPKVQKLD PLLKVKKLS PAzKKxKz SPzKRxRz

The NLS sequence of Drosophila 23 kDa dUTPase shows homology with the NLS of human c-myc and RanBP3 (A). Examined dUTPases from different eukaryotic organisms possess similar NLS as found in Drosophila or human larger isoforms (B). These NLS sequences contain only three basic amino acids at conserved positions, and in many of them, one proline residue is also present. The PAAKKMKID sequence motif is 100% conserved among available Drosophila genomes [25] (data not shown). z, non-charged amino acid; x, any residue.

of NLS class, where a short cluster of basic residues is flanked by neutral and acidic amino acids. The Drosophila dUTPase NLS also resembles the previously described human dUTPase NLS and human RanBP3 NLS as it contains only three positively charged amino acids in the middle of the NLS motif. Comparison of dUTPase sequences from different eukaryotic species has revealed that they possess similar NLS segments at their N termini (Table 1). Localization shifts of Drosophila dUTPase within embryos The S2 cell studies did not offer additional insights to explain the unexpected simultaneous localization of both isoforms in either nuclear or cytoplasmic compartments [13]. In Drosophila embryos at the syncytial blastoderm stage, events of synchronized nuclear cleavages follow each other, providing an excellent system for investigating cellular trafficking during mitotic processes. Such experiments were carried out wherein S2 cell extract, containing the 23 kDa or 21 kDa fluorescent dUTPase constructs, was microinjected into embryos and the localization patterns were followed during nuclear cleavage. Rhodamine–tubulin was also coinjected to aid visualization of the mitotic stages. Steps of mitosis were determined by following the formation and movements of mitotic spindle and centromers, thus localization of dUTPase isoforms could have been timed to mitotic stages. Fig. 2A and B (cf also Supplementary movies 1–4) clearly indicates that during interphase, the 23 kDa isoform staining is located within the nuclear space while the 21 kDa isoform is diffusely scattered and is excluded from the nuclei. In agreement with S2 cell culture studies we observed that presence or absence of NLS clearly distinguishes the two isoforms regarding their localization. As nuclei enter mitosis, the chromatin gets condensed and the intact nuclear envelope disintegrates. While nuclear pore complexes disassemble, the nuclear envelope becomes permeable. Size- and NLS-selective, active nuclear transport process through the pores can not function from prophase [24]. As shown in Fig. 2, starting from this specific stage, the two dUTPase isoforms show opposite localization shifts. Unexpectedly, the 21 kDa dUTPase shows a localization shift to the karyoplasm, meanwhile the

23kDa isoform

21kDa isoform Interphase

Interphase

NLS

dUTPase

dUTPase

NPC

NLS dependent Involves NPCs

nucleus

Prophase

NLS independent NPC independent

NLS dependent

nucleus

dUTPase

dUTPase

NLS independent NPC independent Telophase

Telophase

dUTPase

Lacking NLS it stays in the cytoplasm Prophase

NLS

NLS

NPC

NPC

NPC

dUTPase

NLS independent

Fig. 3. Differential localization mechanisms operate for the two Drosophila dUTPase isoforms.

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23 kDa dUTPase starts to diffuse from the nuclear space. None of the isoforms shows colocalization with the condensated chromosomes. Later on during metaphase, the 21 kDa dUTPase remains around the chromosomes which have aligned at the metaphase plate. Remnants of 23 kDa dUTPase can be detected in dividing nuclei, but most of the 23 kDa dUTPase is scattered all around in the cytoplasm. Localization of dUTPase isoforms does not change much during anaphase; the 21 kDa dUTPase traces out the chromosomes, which are pulled apart towards the opposite poles. The 21 kDa dUTPase gets excluded from the karyoplasm only when new nuclei of the daughter cells and their nuclear envelope emerge. During telophase, nuclear pore complexes also assemble, resuming active and controlled transport into or out from the nuclei. By the end of cytokinesis, embryo regains the state of interphase, when 23 kDa is nuclear and 21 kDa dUTPase is cytoplasmic. Discussion The above described localization shifts of two dUTPase isoforms through the spindle envelope show synchronized character and they are closely timed to the nuclear cleavage phases. Fig. 3 schematically summarizes the observed results. For the NLS-containing 23 kDa isoform, the localization shifts are probably due to an importin-dependent mechanism through the NPC, as these follow the general pattern dictated by the NLS-dependent trafficking. This NLS segment is well preserved in other eukaryotic dUTPase sequences (Table 1) indicating that in most cases, the enzyme can be transported to its physiologically cognate cellular compartment, i.e. the nucleus. In the case of the 21 kDa dUTPase isoform, however, the localization shifts show unexpected pattern that cannot be explained solely on the basis of NLS- and NPC-dependent processes. Data suggest that nuclear localization of both dUTPase isoforms is under strict regulation involving factors beyond the NLS and nuclear pore-based Ran transport system. This behavior explains how dUTPase can be observed within both the cytoplasm and the nuclei [13], although at different stages of mitosis. The present results clearly indicate that the role of dUTPase isoforms in Drosophila is at least partially different from those described in humans with respect to the NLS-lacking isoform. In humans, this isoform is a ‘‘bona fide” mitochondrial protein [14], however, in Drosophila, the second isoform may also be present in the nuclear space, at least in the syncytial stage of embryonic development. During this stage, mitotic cycles follow each other in a very rapid manner. Our present results suggest that the presence of dUTPase in the nuclear space might be of increased importance during the whole cell cycle to provide accurate regulation of dUTP/dTTP pools for both repair and replicative DNA synthesis. Acknowledgments This work was supported by grants from Hungarian Scientific Research Fund (K68229) (OTKA, NI 69180); Howard Hughes Medical Institutes (#55005628 and #55000342); Alexander von Humboldt Foundation; and JÁP_TSZ_071128_TB_INTER from the National Office for Research and Technology, Hungary; FP6 STREP 012127; FP6 SPINE2c LSHG-CT-2006-031220; and TEACH-SG LSSG-CT-2007-037198 from the EU. Appendix A. Supplementary data Confocal images of microinjected Drosophila embryos were recorded for two complete nuclear division cycles. Movies show one cycle for each construct. The fluorescent signal of the respec-

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