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.
25469
25470
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
409
67
4 0 1 CCT ATG CTC TTC CAA CGT AAA GCT GTT CTT CCC T C I GCC C O T T T A ATG A T C GGT G T T ACC
4 9 0 CCA GGT K C M C C M GAA ACA TCT "X ATT GCT ATT AAA grga9LagtttgTLatCL~TCacaLtLf 5 5 6
80
6
8
F
G
T
N
Q
E
R
S
F
I
A
I
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7
9
R
M
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E
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V
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S
A
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n
I
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v
T
462
98
ti09 TTC GCT CTT CAT GTI' GGT AGA M C ATC ATC CAC CGT TCT GAT TCA CTT GAA TCT GCC M C 6 6 0
111
1
1
2
F
C
Y
D
V
C
R
N
l
I
H
C
5
0
S
Y
E
S
A
N
9 3 9 CCTTTC
9
5
R
C M M C AM
F
E
K
CCT TTC A M TTA GTT COT A T T AAA AT:
K
C
F
K
L
V
G
I
K
I
CTT GTT CCA ACT CCA GAA
L
V
P
T
F
E
990
114
C
G M ACT GCC CGT AAT ATT ATC CAC
GGT
X A GAT TCA AAT G M TCA
1 2 5 9 TTC GAT T T A TGTATT
1
1
4
F
D
L
C
I
E
T
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1118
193
Gipl7
1 4 5 2 a ~ d ~ a ~ t ~ ~ ~ . d l ~ g r ~ ~ C L ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . r L L L a t L ~ L C a L L ~ t t1 5=1 1l L ~ t t t ~ ~ ~ t t t ~ ~ t t t t t ~ t t t ~ t t t t ~ ~ ~ ~ = ~
1512 L t a ~ ~ a * l C C a C ~ g g t l l d r ~ ~ C t ~ t ~ ~ ~ ~ t = t t 9 ~ ~ 9 9 ~ 9 ~ ~ 9 ~ 9 9
1511
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
MFSRFARAFPKILASGASQRTFATVQKAFGNPTSKKLIVGSSLLIGSAFATTSFVACENKSVPL
64
VGLPG~QERSFIAIKPDGTQRRLIGEIIARFMKGFKLVG
M S ~ . KT.L.V ....VA.G.V ......Y
V...L.Q....KDL.ES.. R M K E
HAA.K..T..MV....V..G.V.K..E...Q......AL.F?WASK.LLE...A..SA
C..
T........V..G.V....K...Q...R...L.FMQASEDLLKE..V..
KD
MAI..T.SI...NAVAKNV..N.F....AA...I..T.H.HL.V.Q.RGF.AEHDG
MA1 TLSI
L MGV..K..S...E..L.P.A.RLQHLSQAQ.EGF.AVHKA
125
..
..
..
.....
.....
KPFFNGLVKFFSSGAWAMVFEGKDWR~RVLJGATVPSQSAPGTIRFDLCIETG~II
R
G...S.IT..P.........G..ASA.IU..V.N.LA....S..G.K;VDV.....
R...P...NYMN..P..P..W..LN..K...~...N.AD.L.....G.F..QV.....
R
A...KYEM..P.....W..LN..K......E.N.AD.K.....G.F..QV.....
D...E.~..PI.VS.L..ENA.QRH.D.L...N.ANALA..L.A.YADSLTE.GT
R...KD..Q.MI..P..L..L..ENA.LAN.DIn...N.A.A.E....K.FATSIDK.TV
...
...
....
61
58
57
56
56
185
121
118
117
116
116
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 (.).
.....
.....
..
...
...
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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Acknowledgments-We are most indebted to R. Bisson for suggesting that the NH2-terminal extension in Guk is a mitochondrial
presequence, to M.L. Lacombe for oligonucleotide primer MLL
hybridizing with the gip17 gene, and toG. Gerisch for a generous gift
of anti-Dictyostelium porin antibodies. We thank D. Malchow for his
encouragement, most helpful discussions, andconstant support
throughout the course of this work.
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