OF BIOLOGICAL CHEMISTRY THEJOURNAL Q 1993 by The American Society for Bioebemistry and Molecular Biology, Inc.

Vol. 268, No. 34, Isaue of December 5, pp. 25469-25475,1993 Printed in U.S.A.

Separate Nuclear Genes Encode Cytosolic and Mitochondrial Nucleoside Diphosphate Kinasein Dictyostelium discoideum* (Received for publication, May 28, 1993, and in revised form, July 28, 1993)

Heike Troll$#,Thomas WincklerSIl, IoanLascu((,Norbert Muller**, William Saurin$$, Michel VeronlJ,and Rupert MutzelSQQ From the SFakultat fur Biobgie, Uniuersitat Komtanz, 78434 Konstanz, Federal Republic of Germany, the (1 Unite de Biochimie Cellulaire, Centre National de la Recherche Scientifique-URA 1129 and $$Unite de Programmation Moliculaire et Toxicologie GinPtque, Znstitut Pasteur, 75724, Paris Cedex 15, France, and the **Znstitutfur Allgemeine Mikrobwlogie, Uniuersitat Bern, 3012 Bern, Switzerland

We have previously isolated cDNA clones for the development (Dearolf et al., 1988) encodes an NDP kinase gip17 gene encoding the cytosolic nucleoside diphos- (Biggs et al.,1990). The human gene nm23-HI which is highly phate (NDP) kinase from Dictyoetelium discoideum, expressed in proliferating malignant cells (Keim et al., 1992; and partial cDNAs for guk, a second member of the Hailat et al., 1991; Lacombe et al., 1991) and which may be NDP kinase gene family (Wallet,V., Mutzel, R., Troll, involved in tumor metastasis (Rosengard et al., 1989; Leone H., Barzu, O., Wurster, B., Veron, M., and Lacombe, et al., 1991) also encodes an NDP kinase (Gilles et al.,1991). M. L. (1990)J. Nutl. Cancer Inst. 80, 1199-1202). Both Awd, Nm23-H1, and asecond isozyme encoded by nm23We now characterize genomic DNA clones for both H2 are highly similar to agene encoding an NDPkinase from NDP kinase genes, and we show that guk defines a the cellular slime mold Dictyostelium discoideum (Wallet et nuclear-encoded mitochondrial NDP kinase. Isolated al.,1990; Gilles et al.,1991). D. discoideum mitochondria contain 3% of the total NDP kinases have been reported to be associated with cellular NDPkinase activity. Antibodies whichspecifically recognize and inhibit the activity of either cyto- subcellular structures or protein complexes, and a number of solic or mitochondrial NDPkinase unambiguously dis- regulatory roles have accordingly been suggested. NDP tinguish between these activities. The nascent mito- kinases should indeed contribute to the regulation of any chondrial NDP kinase contains a presequence of 67 cellular process requiring non-adenine NTPs since they obviously sit at acentral node in cellular metabolism. The amino acids that is removed during import into the organelle as shown by determination of the NH2 ter- presence of membrane-associated NDP kinase activity (Kiminus of the mature protein from mitochondria. The mura and Shimada, 1988; Wieland et al.,1991) and its ability genes formitochondrialandcytosolicNDP kinases to synthesize GTP has led to a proposed role in the regulation contain four and two introns, respectively. The posi- of GTP-binding proteins (reviewed in Lacombe and Jakobs, tions of the introns in gene the for the cytosolic enzyme1992). NDP kinases were also found associated with micromatch exactly the positions of the second and fourth tubules (Nikerson and Wells, 1983; Biggs et al., 1990); their introns in the coding region of its mitochondrial hom- role in microtubule polymerization is, however, not yet clear ologue. From these results we conclude that the isosince no effect on the kinetics of this process in vitro could zymes diverged from acommon ancestor, and we dis- be demonstrated (Melki et al.,1992). cum possible phylogenetic pathways for the evolution Because of their ability to synthesize deoxytriphosphonuof cytosolic and organelle NDP kinases. cleotides, NDP kinases also are candidates to participate in multi-protein complexes of the DNA replication machinery. NDP kinase is present in highly purified bacterial DNA Nucleoside diphosphate (NDP)’ kinases (EC 2.7.4.6) are polymerase preparations (Miller and Wells, 1971) and has ubiquitous enzymes that transfer they-phosphate from ATP been reported associated with ribonucleotide reductase (Allen to diphosphoribonucleosidesand deoxyribonucleosides (Parks et al., 1983). Another possible target for the action of NDP and Agarwal, 1973). In Drosophila the awd gene involved in kinases is the cellular pteridine metabolism; the developmental Drosophila mutant Killer of prune, a non-lethal allele * This work was supported by the Deutscher Akademischer Aus- of awd (Biggs et al.,1988),shows a dominant lethal interaction tauschdienst/CNRS Procope program, the Deutsche Forschungsgemeinschaft, SFB156, INSERM 920113, and the Lime de la Recherche with the prune mutant (Sturtevant, 1956) which wasrecently Contre le Cancer, Comiti de Paris. The costs of publication of this demonstrated to drastically lower the concentrations of larval article were defrayed in part by the payment of page charges. This pteridines (Hackstein, 1992). article must therefore be hereby marked “aduertisemnt” in accordThe existence of a mitochondrial NDP kinase activity has ance with 18 U.S.C.Section 1734 solelyto indicate this fact. been reported as early as 1955 (Herbert et al., 1955). In rat The nucleotide sequence(s1 reported in this paperhas been submitted to the GenBankm/EMBL DataBank with accession number(s) L23067 liver the enzyme is located in the intermembrane space, where it could couple respiratory chain-driven ATP production diand L23068. rectly to the formation of nonadenine nucleoside triphos§ Supported by a fellowship from the Claussen Stiftung. ll Present address: Institut fiir pharmazeutische Biologie, Univer- phates (Pedersen, 1973). This view is supported by more sitiit Frankfurt, Georg-Voigt-Str. 16,60325 Frankfurt am Main. FRG. recent observations demonstrating that mitochondrial §§ To whom correspondence should be addressed. Tel.: 49-7531kinases of the intermembrane space act in concert with the 882479; Fax: 49-7531-882966. The abbreviations used are: NDP, nucleoside diphosphate; NTP, adenine nucleotide translocase (Brdiczka, 1991). Mitochonnucleoside triphosphate; cNDK, cytosolic NDP kinase; mNDK, mi- drial NDP kinase could thus constitute an important link tochondrial NDP kinase. between energy metabolism and cellular regulation.

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Mitochondrial Dictyostelium NDP in Kinase

The amino acid sequences of several prokaryotic and cytosolic eukaryotic NDP kinases have recently been reported (Munoz-Dorado et al., 1990; Lacombe et al., 1990; Kimura e t ai., 1990; Gilles et aL, 1991; Hama et aL, 1991). Their sizes vary very little with extremes of 143 residues for the Escherichia coli, and 155 for the Dictyostelium enzyme. The E. coli and human enzymes share 43% identity (Hama et al., 1991), and 60-65% sequence identity is found between mammalian NDP kinases and Dictyostelium NDP kinase (Wallet e t al., 1990). The existence of cytosolic NDP kinase isozymes in mammalian tissues has been known for a long time (Parks and Agarwal, 1973), and it has recently been demonstrated that thedifferent human isozymes are made of only two types of subunits which combine to form hybrid hexamers (Gilles et al., 1991). So far, no primary structures of mitochondrial NDP kinases are available, and it unclear is whether the NDP kinase activity associated with mitochondria is encoded by a different gene than thecytosolic enzymes. We have previously isolated cDNA clones for the cytosolic NDP kinase (Gipl7) from D. discoideurn (Lacombe e t al., 1990; Wallet et al., 1990). The enzyme was subsequently purified from recombinant bacteria and itsthree-dimensional structure determined at 2.2 A resolution by x-ray diffraction (Dumas et al., 1992). Isolation of partial cDNA clones for a homologue (Guk) of this protein (Walletet al., 1990)indicated the presence of a second member of the NDP kinase family in thecellular slime mold. In order to analyze thenatureand possible functional significance of this second NDP kinase gene, we have now determined its structure, and we show that it encodes the mitochondrial NDP kinase from D.discoideum. Specific antibodies were used to formally discriminate between the cytosolic and mitochondrial isozymes. A phylogenetic analysis leads us to conclude that the genes for the cytosolic and organelle enzymes diverged from a common ancestor and provides insights into possible evolutionary pathways for the NDP kinase gene family. EXPERIMENTAL PROCEDURES

Materials-Colony/Plaque Screen discs and GeneScreen hybridization transfer membranes were from DuPont-New England Nuclear, and BA85 nitrocellulose filters were from Schleicher & Schuell. Immobilon membranes were from Millipore. Restriction enzymes and DNA-modifyingenzymes were obtained from Boehringer Mannheim, Life Technologies, Inc./Bethesda Research Laboratories, and New England Biolabs. T7 gene 6 exonuclease was from United States Biochemical Corporation, and the “Cyclone I Biosystem” was from International Biotechnologies,Inc. The “double-strandednested deletion kit” was supplied by Pharmacia. [y3*P]ATP(3000 Ci/mmol), [a-32P]dATP(3000 Ci/mmol), [a-%]dATP (>400 Ci/mmol), an M13 sequencing kit, and the ”ECL” chemiluminescence detection kit for Western blotting were from Amersham. The multiprime DNA labeling system used for the synthesis of DNA probes was from Life Technologies Inc./Bethesda Research Laboratories. Peroxidase-conjugated anti-rabbit antibodies were purchased from Dianova, Hamburg, Federal Republic of Germany. Oligonucleotides were from MWG-Biotech, Ebersberg, F.R.G., and from the Uniti de Chimie Organique, Institut Pasteur. Phages and Bacterial Strains-The Xgtll cDNA library described by Lacombe et al. (1986) was used.The E. coli SURE strainwas from Stratagene.Other vectors and bacterial strainsare described in (Sambrook et al., 1989). Oligonucleotides-Oligonucleotides used for the determination of the guk sequence were as follows: HT1, 5”GCGATTTCATHT3, GAGCAGCAG-3’; HT2, 5’-CCATAGCAACAACAGCACC-3’; 5‘-CTTGGGTACCATCTGG-3’; HT4, 5”GTGGTGGCAAAGGCGGAACC-3‘; HT5,5’-CGTTGAGAAGCACCTGAGGC-3’. Oligonucleotides hybridizing with the gip17 gene were: MLL, 5’-GCGATGATTTCACCAAC-3’; 101,5’-CCTCTGGTCCAGTCGTTGC-3’; 105,5”GAAACATCATCCACGGTTCTGATTC-3’; 106, 5‘-CATCGCCAGATACGAAAAGAAAGG-3‘; 107,5-C’l”AGCTGA-

ATCTCACTATGCTGAA-3’; 5”GTGTTCAGCATAGTGAG-3’. Construction of a GenomicD. discoideum Library in pUCl9Genomic DNA was prepared from nuclei of D. discoideum strain Ax2 as described (Welker et al., 1985). 5 pg of DNA was digested with HincII and ligated with 5 pg of HincII-digested pUC19 plasmid DNA. E. coli SURE cells were transformed with the ligation mixture by electroporation (Sambrook et al., 1989). Screening of the Agtll cDNA Library and pUCl9Genomic Library with cDNAProbes-Probes were synthesized by hexanucleotide priming (Feinberg and Vogelstein, 1983) using the -0.4-kilobase guk7.2 cDNA or the gipl7 cDNA (Wallet et al., 1990) as templates. The guk7.2 cDNA corresponds to about 60% of the open reading frame in the guk gene. A total of 3.5 X lo5 phages (lO‘/plate) from the Xgtll library were plated on Y1088 (Young and Davis, 1983), transferred to Colony/Plaque Screen discs, and hybridized with the guk cDNA probe according to the protocol described by the manufacturer. For isolation of genomic clones 3 X lo‘ colonies from the genomic library in pUC19 (5 X IO3 colonies/plate) were screened using the same protocol. DNA Sequeming-cDNA inserts from Xgtll clones were subcloned into M13mp19. Single-stranded DNA was prepared following standard protocols (Sambrook et al., 1989), and processive deletions were prepared according to the technique of Dale et al. (1985). DNA sequence analysis was performed according to Sanger et al. (1977) modified for use with [a-%]dATP (Biggin et al., 1983). Processive deletions of genomicDNA inserts were prepared by exonuclease treatment using the double-stranded nested deletion kit following the protocol provided by the manufacturers. Double-stranded DNA sequencing was performed on plasmid DNA purified by CsC1-gradient centrifugation. For sequence determination with internal oligonucleotide primers, the recombinant pUC19 DNAwas digested with HindIII and treated with T7 gene 6 exonuclease to produce two single-stranded halfs of the plasmid which were used as sequencing templates. DNA and protein sequences were analyzed using the DNA strider program (Marck, 1988). Preparation of Dictyostelium Mitochondria-Mitochondria from vegetative D. discoideum cells were prepared by differential centrifugation as described (Troll et al., 1992); briefly, 2 X lo* cells/ml suspended in STEB buffer (20 mM Tris-HC1, 1 mM EGTA, 0.2 M sucrose, 0.2% w/v bovine serum albumin, pH 7.6) were disrupted in a Waring Blendor and the crude extract was centrifuged for 20 min a t 27,000 X g. The pellet was resuspended in the original volume of STEB buffer; cell debris, unlysed cells, and nuclei were removed by centrifugation (5 min, 480 X g), and themitochondria were collected by centrifugation for 10 min at 16,000 X g and resuspended a t 1/20 of the original volume in STEB buffer. Immunological Experiments-The antiserum used for screening of the Xgtll expression library, termed anti-Guk, has been previously described (Wanner and Wurster, 1990; Wallet et al., 1990). It allowed isolation of cDNA clones for both genes gipl7 andguk (Wallet et al., 1990). It recognized non-denatured Gipl7 protein during immunoscreening and in enzyme-linked immunoadsorbant assays, but it did not cross-react with Gipl7 after transfer from SDS-polyacrylamide gels to nitrocellulose. For Western blotting experiments, anti-Guk antiserum was affinity-purified on the h2-guk10.3-encoded fusion protein expressed in a Y1089 lysogen (Young and Davis, 1983) according to thetechnique described by Goldstein et al. (1986). Soluble NDP kinase was detected with a polyclonal antiserum (anti-Gipl7) raised against recombinant Gipl7 (Wallet et al., 1990). This serum inhibits the catalytic activity of Gipl7. It did not cross-react with the lacZ-cDNA-encoded fusion proteins of cDNA clones guk7.2 and guk10.3. Inhibition of soluble and mitochondrial NDP kinases by the antisera was assayed after preincubation of enzyme fractions with antibodies for 30 min at room temperature. Mitochondria were treated with 1%Triton X-100 prior to use for the inhibition assay. Incubation with preimmune sera orwithout added antiserum showed no decrease of the enzyme activities during the preincubation period. Partial Purification of Mitochondrial NDP Kinase and Determination of Its NH, Terminus-Mitochondria were lysed by sonication, was applied centrifuged for 10 min a t 10,000 x g, and the supernatant to a DEAE-Sephacel column equilibrated with 50 mM Tris acetate, pH 8.0, 1 mM EDTA. The NDP kinase activity, recovered in the flow-through, was precipitated by dialysis against a saturated solution of ammonium sulfate. The precipitate was dissolved in a small volume of 50 mM Tris acetate, pH 8.0, 1mM EDTA, 0.5 M NaCI, clarified by centrifugation for 10min at 10,000 X g, and subjected to gel filtration on a Sephacryl S-200 column. Fractions were analyzed for NDP

MitochondrialNDP Kinase in Dictyostelium

25471

kinaseactivityandcross-reaction with anti-Gukantibodies.The gion encoding amino acids 13-20 in the guk open reading fraction containing most of the mitochondrial NDP kinase activity frame, recognized a transcript from Dictyostelium cells that waschromatographed on a 15% polyacrylamide, 0.1% SDS gel. A major band migrating at 17 kDa was transferred to an Immobilon was indistinguishable both in size and developmental regulation from the guk mRNA (data not shown). Close inspection membrane and subjected to microsequencing. Phylogenetic Tree Reconstruction-NDP kinase primary sequences of this amino-terminal extension revealed that it is structurfrom the following sources were extracted from the SwissProt protein ally similar to the bipartite presequences of nuclear-encoded sequencelibrary: E. coli(Hamaet al., 19911, Myxocoecrcsxanthrcs proteins that are targeted into the mitochondrial interrnem(Munoz-Doradoet al., 19901, Drosophila melanogaster (awd, Biggs et brane space (see Pfanner and Neupert, 1990, for review). al., 1988), Ginglymstoma cirraturn (Kasahara et al., 1993), mouse To demonstrate that guk encodes a mitochondrial enzyme, (Urano et al., 1992), rat (Kimura et al., 1990; Shimada et al., 1993), and human (Rosengard etal., 1989). To calculate distances between mitochondria from Dictyosteliumwere separated from the cytosol by a differential centrifugation technique (Troll et al., pairs of sequences, the columns of the global alignment including gaps were excluded. The distancewas then calculated as the percent 1992), and both fractionswere assayed for NDP kinase activof amino acid differences between sequences. The phylogenetic tree ity and immunological cross-reaction with anti-Gipl7 and was computed according to the unweighted pair-group method with anti-Guk antibodies. Activity of the mitochondrial NDP kiarithmetic mean method (Sokal and Michener, 1958). nase preparation amounted to 3.2 units/g of cellular protein, Other Methods-SDS-polyacrylamide gel electrophoresis and Western blotting experiments were performed as described (Mutzel about 3% of cytosolic activity (82.6 units/g), a value compaet al., 1988). Peroxidase-conjugated secondary antibodies were de- rable with published data from mammalian organisms (Parks tected with the ECL chemiluminescence staining procedure according and Agarwal, 1973). The measurable mitochondrial NDP to the manufacturer's instructions. Protein concentrations were de- kinase activity was identical in fresh preparations and after termined according to Bradford(1976) with bovineserum albumin as lysis of the organelle by 1%Triton X-100 (not shown), india standard. NDP kinase activity was measured as described (Parks andAgarwal,1973)usingdTDP as a substrate. 1 unit of enzyme cating that the enzyme is located in a compartment that is freely accessible to itssubstrates. transfers 1 pmol of phosphate/min to dTDP. Western blots from the fractions probed either with antiGipl7 or affinity-purified anti-Guk antibodies and monoRESULTS clonal antibodies against Dictyostelium porin (Troll et al., Structure of the guk and gipl7Genes-In order to obtain a 1992) showed that Gipl7 was almost exclusively found in the full-length cDNA clone for guk, 3.5 X 10' phages of the cDNA cytosol (Fig. 3A). In contrast, Gukwas highly enriched in library were rescreened with the guk7.2 cDNA as a probe. mitochondria (Fig. 3B). It migrated at the same apparent The longest cDNA clone isolated (guk 10.3) encoded a protein molecular mass as Gipl7 (17 kDa) suggesting that the NH2of 218 amino acids, but did not contain the initiator ATG terminal presequence is cleaved during import into the mitocodon (not shown). chondria (see below). The cDNA probe was therefore used to screen a genomic The identity of the guk gene product as a mitochondrial library constructed by ligation of Dictyostelium DNAinto the NDP kinase is further established by using specific antibodies Him11 site of plasmid pUC19. A 3.3-kilobase genomic clone to inhibit the NDPkinase activity in cytosolic and mitochonwas isolated. Fig. 1A shows the nucleotide sequence of the drial fractions. Soluble NDP kinase activity could effectively gene and thepredicted primary structureof the Guk polypep- be inhibited with anti-Gipl7 antibodies but was unaffected tide. Note that cDNA clone guk10.3 encoded almost the entire by preincubation with anti-Guk antiserum (Fig. 4A). In concoding region, lacking only the 5' five nucleotides. Guk contrast, preincubation of the mitochondrial NDP kinase with tains four introns of 83-237 base pairs in length, displaying a anti-Guk antiserum resulted in an over 80% inhibition of the more complex structure than most Dictyostelium genes; inactivity, whereas anti-Gipl7 antibodies had only a minor trons I1 and I11 are separated by only seven nucleotides of effect (Fig. 4B). In eithercase, the combined addition of both coding sequence, one of the smallest exons so far discovered antisera did not significantly reduce the activity further. in Dictyostelium genes. Biochemical Properties of the Dictyostelium Mitochondrial Screening of the genomic DNA library yielded two additional clones hybridizing only weakly with the guk7.2 probe NDP Kinase-NDP kinases can undergo autophosphorylabut strongly with a gipl7 cDNA probe (not shown). Sequenc- tion on a histidine residue in the presence of a nucleoside ing of one of these clones showed that they indeed corre- triphosphate and MgClz, as partof their reaction mechanism sponded to gipl7(Fig. 1B). Gipl7contains two introns of 144 (Parks and Agarwal, 1973). Autophosphorylation was also and 107 base pairs. Their positions in thecoding region match found for the Dictyostelium mitochondrial NDP kinase: a 32Pexactly the positions of introns I1 and IV in guk. Fig. 1C labeled 17-kDa product was detected after SDS-polyacrylsummarizes the physical organization of genes guk and gipl7. amide gel electrophoresis of the mitochondrial fraction upon Guk Is a Mitochondrial NDP Kinase-The sequence from incubation in the presence of 1 mM MgC12 and [y3'P]ATP residue 71 in Guk to thecarboxyl terminus is similar to NDP (not shown). The structural analogy between the NH2-terminal extenkinases from both prokaryotic and eukaryotic organisms (Fig. 2). 40 out of 143 residues in this region are identical in all sion in the guk gene product with the presequences of mitoNDP kinases and in Guk. This includes a ubiquitously con- chondrial enzymes and the difference in molecular weight between the predicted guk product and the mitochondrial served histidine residue corresponding to His''' inGipl7 which is autophosphorylated during the ping-pong phospho- protein (Fig. 3) indicated the occurrence of a processing of transferase reaction (Gilles et al., 1991; Dumas et al., 1992). the precursor protein. In order to identify the cleavage site, Homology of Guk with Gipl7 andwith other prokaryotic and the mature protein was partially purified from Dictyostelium eukaryotic NDP kinases is discussed below (see Discussion). mitochondria by ion-exchange chromatography and gel filtraA striking feature of this sequence comparison is the NH2- tion, and its NH2 terminus determined by microsequencing. terminal extension found in Guk, resulting in a predicted The sequence found was NH2-Glu-Asn-Lys-Ser-Val-Pro-Leu. polypeptide of 220 residues as compared to 155 in Gipl7.This This identifies the cleavage point of the presequence at posiextension does not reflect a cloning artifact since it isencoded tion 57 and suggests an import mechanism similar to thatfor both by cDNA clone gukl0.3and by the genomicDNA. other nuclear-encoded proteins of the mitochondrial interFurthermore, oligonucleotide HT5, complementary to the re- membrane space (Pfanner and Neupert, 1990). The matura-

25472

Mitochondrial NDP Kinase in Dictyostelium

4 3 0 CCC TIT GCC ACC ACT TCT TIT CTT GCI TCT G M AAT AAA WA CTC C C I TTA GTT CCT CTT 4 8 A F A T T S F V A C E N K S Y F L Y G L

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FIG. 1. DNA sequences, deduced amino acid sequences, and comparison of the structures of genes guk and g i p l 7 . See "Experimental Procedures" for experimental details. Coding regions are shown in upper case letters and nontranslated sequences in lower c u e letters. A, sequence of guk. The 5' ends of cDNA clones guk7.2 and guk10.3 are located at positions 1014 and 186, respectively. B, sequence of gipl7. C,comparison of the structures of guk and gip17; open boxes represent exons, shaded bores introns (numbered in roman letters), and solid lines 3' and 5"nontranslated regions, respectively. The borders of common introns in guk and gipl7 are marked.

GUK

GUK GIP17 AWD NM23-H1 E. COLI

n.xmmms GUK GIP17 AWD NM23-Hl E. COLI W.XANTHU.5

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GUK HGSDSNGSAAHEIALMFKEDEIANWVSTNPVYEKM 220 GIP17 .....VE. .NR.......PE.LLTE.KP..NLYE 155 AWD ....AVE..EK ......NEK.LVT.TPAAKDWIYE 153 NM23 -H1 VE ..EK G...HPE.LVDYT.CAQNWIYE 152 E. COLI VE...R YF.GPG.VCPRTR 143 M.XANTHUS .....LEN.KI YF.RPT..HSYPYQK 145 FIG. 2. Alignment of the predicted amino acid sequence of the Dictyoetelium Guk protein with the amino acid sequences of prokaryotic and eukaryotic cytosolic NDP kinases. For clarity, only the human nm23-HI gene product is shown as a typical example for the mammalian enzyme. Sequence identities of guk with any of the other proteins areindicated by (.).

..... .....

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tion process thus leads to a mitochondrialNDP kinase similar in size to thecytosolic enzymes (see Fig. 2). The mitochondrial NDP kinase activity eluted at thesame exclusion volume as Gipl7 upon gel filtration (not shown), indicating that theholoenzyme is hexameric like its cytosolic homologues (Dumas et al., 1992).

The thermalstability of cytosolic and mitochondrial NDP kinases was investigated. Half-maximal inactivation of the mitochondrial activity occurred at 47.5 "C,whereas the cytosolic activity was more stable, half-maximal inactivation being observed at 58 "C (data not shown). We speculate that the lower stability of the mitochondrial NDP kinase may be

Mitochondrial NDP Kinase in Dictyostelium

25473

which is structurally analogous to the mitochondrial import sequences of proteins directed into the mitochondrial intermembrane space. This signal sequence is removed by cleavage 88 during import into theorganelle, resulting in apolypeptide of 56 only 6 residues longer than the cytosolic Dictyostelium en38.5 33.5 zyme, and 9-10 residues longer than that of other cytosolic NDP kinases. The genes (and encoded proteins) of the two Dictyostelium 12 members of the NDP kinase family had previously been termedgipl7 (Gipl7) andguk (Guk). We now designate ndkC 1 2 3 14 25 3 4 5 1 2 3 4 5 (cNDK) the gene for the cytosolic enzyme (and its encoded FIG. 3. Guk is localized in Dictyostelium mitochondria. A product) and ndkM (mNDK) thegene for the mitochondrial activity (and itsencoded product), respectively. crude extract was fractionated as described under“Experimental Procedures.” Aliquots of the fractions were chromatographed on 15% Comparison of the cytosolic and mitochondrial NDP k‘mase polyacrylamide, 0.1% SDS gels, the proteins transferred to nitrocel- genes from the cellular slime mold provides insight into the lulose and subjected to Westernblotting with anti-Gipl7 (A) or evolution of the enzyme family. A phylogenetic analysis of affinity-purified anti-Guk ( B ) antibodies. C shows Western blots with anti-Dictyosteliurn porin antibodies as a mitochondrial marker representative NDP kinase primary structures (Fig. 5) shows that the Dictyostelium genes diverged very early from other (Troll et al., 1992). To the left, top and front of the gels and the positions and sizes (in kDa) of molecular mass marker proteins are eukaryotic NDP kinases, in agreement with previous data on indicated. Lanes: 1, crude extract; 2,27,000 X g supernatant; 3,27,000 the molecular phylogeny of cellular slime molds (Sogin et dl., X g pellet; 4, 16,000 X g supernatant of the washed pellet; 5, mito1989). The genes for mitochondrial and cytosolic NDP kinase chondrial fraction. 50 pg of protein was applied in each lane. have diverged further than any pair of eukaryotic cytosolic NDP kinases analyzed so far. cNDK is the counterpart for plant and animal cytosolic NDP kinases. The subunit isoB forms identified in mouse, rat, andhuman cells probably arose late by duplication. The considerable degree of sequence divergence between Dictyostelium cNDK and mNDK also predicts arelatively strong sequence divergence between cytosolic and mitochondrial NDP kinasesin other organisms. This would explain why mammalian or insect genes for mitochondrial NDP kinases were not detected by low stringency hybridization using DNA probes for the cytosolic enzymes. Indeed, in Dictyostelium and Drosophila only single genes for cytosolic NDP kinase were detected2 (Biggs et al., 1990; Lascu et d.,1992): Recently, the sequences of both the rat NDPkinase LY and /3 genes encoding the homologues of the human cytosolic B and A isoforms, respectively, have been reported (Ishikawa et al., 1992; Shimada et al., 1993). Both genes contain four introns a t identical positions inthe coding regions. However, the positions of these intronsdo not match any of the introns .-n in ndkC or ndkM. The rat LY and /3 genes arearrayedin Y ..-.. + tandem, only about 3 kilobases apart from each other, indiC m m cating again that thegene duplication leading to thecreation FIG.4. Inhibition of cytosolic and mitochondrial NDP ki- of the two mammalian cytosolic isoenzymes has been a recent n e activity by anti-Gipl7 and anti-Guk antibodies. 40-pl aliquots of 10-folddiluted cytosol (0.81 mg/ml of protein, panel A ) or evolutionary event. Conservation of the intron-exon strucmitochondria (1.4 mg/ml of protein, panel B ) were incubated for 30 tures in the genes for rat cytosolic NDP kinase is thus not min a t room temperature with 4 pl of anti Gipl7 or anti-Guk anti- surprising. The differences in the physical organization of bodies or with 8 pl of a mixture of both sera in a total volume of 50 ndkC and the rat genes indicate that remodeling of the intronpl, and theNDP kinase activity was then compared to thatof controls exon structure has also occurred after the genes for cytosolic preincubated without antiserum. 100% activity corresponded to 1.15 and organelle NDP kinases diverged. It is worth noting that units/mg for the cytosolic and 0.35 unit/mg for the mitochondrial none of the introns so far discovered in an NDP kinase gene fractions, respectively. interrupts the coding information for a secondary structure motif identified in the cNDK protein (Dumaset al., 1992). significant in view of the import of the nascent protein into NDP kinases are well-conserved from eubacteria to eukarmitochondria because it could facilitatemaintenance of a yotes. The precise conservation of two introns in the Dictyospartially unfolded precursor duringthe targeting process. telium ndkC and ndkM genes (Fig. 1)shows that these genes share a common ancestor. Two evolutionary pathways for DISCUSSION eukaryotic NDP kinases are possible. The gene encoding the During the last3 years, a number of highly similar primary mitochondrial enzyme could arise by duplication of the gene structuzes of NDP kinases both from prokaryotic and eukar- encoding the cytosolic enzyme, or, alternatively, it could origyotic organisms have been determined (see Introduction). We inate from the genome of the endosymbiont ancestor to the now report the isolation of a Dictyostelium gene encoding a mitochondria and be transferred to the host genome theremitochondrial NDP kinase, the first mitochondrial member after. It is not yet possible to discriminate between these of this enzyme family that hasbecome accessible to molecular analysis. The mitochondrial NDP kinase is nuclear-encoded H. Troll and R. Mutzel, unpublished observation. and contains an NH2-terminal presequence of 57 amino acids A. Shearn, personal communication.

A

B

C

Mitochondrial NDP Kinase in Dictyostelium

25474

24

E. coli

5 24

M. xanthus 20

8

cNDK, Dictyostelium

18

mNDK, Dictyostelium

2

-

12

Awd, Drosophila 10

7

Fish (Ginglymostoma cirratum)

%

Mouse B Human B (Nm2341)

Rat alpha Human A (Nm23-HZ) Rat beta

2

2

Mouse A FIG. 5. Rooted phylogenetic tree of NDP kinase primary structures. The tree was reconstructed using the UPGMA method (see “Experimental Procedures”). The numbers above each branch (horizontal lines) represent distances. Vertical lines indicate the positions of common ancestors; their length is not significant. The difference of topology in the two sets of mammalian sequences suggests that distances less or equal to 2 are not significant.

possibilities since we cannot decide unambiguously which of the Dictyostelium genes is more closely related to the eubacterial NDP kinase genes (see Fig. 5). The presence of conserved introns in nuclear genes encoding mitochondrial or chloroplast enzymes and their cytosolic homologues has previously been taken as evidence for the presence of introns in the genome of the progenotic organism that became the mitochondrial endosymbiont (see Iwabe et al., 1990, and references therein). It will be important to determine the NDP kinase primary structures from the organelles of other eukaryotes. The mitochondrial NDP kinase accounts for only 2-3% of the measurable in vitro NDP kinase activity of total cell extracts. However, since it is located ina small cellular compartment where the concentration of itssubstrates is expected to be fairly high, it might play an important role in cellular metabolism and regulation. Genetic evidence in support of this view comes from Drosophila larvae homozygous for an awd deletion which have 2% of the wild-type NDP kinase activity (Biggs et al., 1990). Since the awd mutation has developmental defects only at thelate third instar larvae (Dearolf et al., 1988) it is possible that the intensive cell proliferation occurring in theearly steps of larval development relies on mitochondrial NDP kinase for NTP and dNTP synthesis. The fact that these compounds would be synthesized in mitochondria should not be a problem for cytosolic delivery since they can freely diffuse through the mitochondrial outer membrane.

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