©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Point Mutations in awd Which Revert the Prune/Killer of Prune Lethal Interaction Affect Conserved Residues That Are Involved in Nucleoside Diphosphate Kinase Substrate Binding and Catalysis (*)

(Received for publication, April 27, 1995; and in revised form, July 20, 1995)

Lisa Timmons Jing Xu Grafton Hersperger Xiao-Fang Deng Allen Shearn (§)

From the Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The awd gene of Drosophila melanogaster encodes a nucleoside diphosphate kinase. Killer of prune (Kpn) is a mutation in the awd gene which substitutes Ser for Pro at position 97 and causes dominant lethality in individuals that do not have a functional prune gene. This lethality is not due to an inadequate amount of nucleoside diphosphate (NDP) kinase activity. In order to understand why the prune/Killer of prune combination is lethal, even in the presence of an adequate NDP kinase specific activity level, and to understand the biochemical basis for the conditional lethality of the awd mutation, we generated second site mutations which revert this lethal interaction. All of the 12 revertants we recovered are second site mutations of the awd gene. Three revertants have deletions of the awd protein coding region. Two revertants have substitutions of the initiator methionine and do not accumulate KPN protein. Seven revertants have amino acid substitutions of conserved residues that are likely to affect the active site: five of these have no enzymatic activity and two have a very low level of specific activity. These data suggest that an altered NDP kinase activity is involved in the mechanism underlying the conditional lethality of the awd mutation.


INTRODUCTION

Nucleoside diphosphate kinases (NDP^1 kinase; EC 2.7.4.6) are ubiquitous enzymes that catalyze the formation of nucleoside triphosphates and maintain equilibrium between nucleoside triphosphate and nucleoside diphosphate pools. Cytosolic and mitochondrial NDP kinases have been purified from a variety of tissue sources(1) . cDNAs encoding several of these NDP kinases have been cloned and sequenced. In some species two distinct cytosolic NDP kinases have been cloned: human nm23-H1 and nm23-H2(2, 3) , rat alpha and beta NDPK(4, 5) , mouse nm23-M1(6) and nm23-M2(7) , and spinach NDPK-I(8) and NDPK-II(9) . The two cytosolic NDP kinases in each species are similar in amino acid composition; the products of the two human genes, NDPK-A and NDPK-B, are 88% identical in sequence, yet each has distinct apparent sizes on SDS-PAGE gels and/or different isoelectric points. In Drosophila, cytosolic NDP kinase is encoded by the abnormal wing discs (awd) gene ((20) ; GenBank(TM) accession number Y00226). The AWD protein is 78% identical to each of the human NDP kinases, NDPK-A and NDPK-B. All NDP kinases exist as multimers. The x-ray structure of the Myxococcus enzyme indicates that it exists as a tetramer(11) , whereas the x-ray structures of Dictyostelium(12) , and Drosophila enzymes(13) , indicate that these enzymes form hexamers. Because Drosophila has only one NDP kinase gene, the AWD/NDP kinase associates into homohexamers, whereas the two human NDP kinases, NDPK-A and NDPK-B, form mixed hexamers(14) .

NDP kinases act via a ping-pong mechanism. The -phosphate of a nucleoside triphosphate is first transferred to the active site histidine of the enzyme(15) , forming an enzyme intermediate. The -phosphate is then transferred from the enzyme to a nucleoside diphosphate acceptor. It has been known for many years that NDP kinases show little specificity toward the sugar or base residue of nucleoside substrates. NDP kinases act upon purine or pyrimidine ribonucleoside diphosphates or deoxyribonucleoside diphosphates(1) . Recent crystallographic evidence has provided an explanation for this broad range of substrate specificity. Unlike the nucleotide binding sites found in GTP-binding proteins (16, 17) and other nucleotide-binding proteins, NDP kinases have no glycine-rich phosphate binding loop or ``Rossmann fold''(18) , and the enzyme has few direct contacts with the base. The nucleotide substrate enters the active site, phosphate first. The active site histidine is in a cleft, and conserved residues lining the cleft primarily interact with the phosphate and ribose groups of nucleotide substrates(11, 19) . This active site configuration is conserved among the enzymes examined so far. Even though the Myxococcus enzyme forms a tetramer and the Drosophila and Dictyostelium enzymes form hexamers, the ADP binding site of the Myxococcus enzyme (11) is remarkably similar to the ADP binding site of Dictyostelium NDP kinase (19) and Drosophila NDP kinase(13) .

The Killer of prune (Kpn) mutation of Drosophila is a point mutation in the awd gene (20) resulting in a substitution of proline at position 97 for serine ((21) ; this paper). KPN/NDP kinase is enzymatically active in extracts from awd individuals and the awd mutation causes no phenotype by itself. The prune gene of Drosophila has been cloned but the biochemical function of its product is unknown(22, 23) . Individuals that cannot make a functional prune gene product are viable, but they have prune-colored eyes rather than red-colored eyes due to decreased amounts of red drosopterin pigments(24) . In the absence of a functional prune gene product, the awd mutation is lethal. Furthermore, only one copy of the mutant awd gene is required for lethality of prune flies(25) . The cause of this lethality is unknown. Lascu et al.(21) found that purified KPN/NDP kinase is less thermostable at 56 °C than purified wild-type AWD/NDP kinase and suggested that in awdmutant larvae an accumulation of KPN monomers occurs. The Pro residue that is altered by the awd mutation occurs in the Kpn loop, a structure that is conserved in all NDP kinases(11, 12, 13) . In Dictyostelium(12) and in Drosophila(13) the hexamer can be divided into a ``top'' trimer and a ``bottom'' trimer. The three Kpn loops from the top trimer are in close proximity on the top of the hexamer as are the three Kpn loops on the bottom of the hexamer. Part of the Kpn loop is thus found at trimer subunit interfaces and may play a role in hexamer stabilization. This structural information provides an explanation for the reduced thermostability of KPN/NDP kinase purified from awd mutant flies and its reduced ability to renature after urea treatment(21) .

The Kpn loops of Myxococcus, Drosophila and Dictyostelium have some residues in the active site of the molecule. For example, Arg of Drosophila and the corresponding Arg of Dictyostelium and Arg of Myxococcus are within the Kpn loop and form hydrogen bonds with the beta phosphate of ADP(11, 12, 13) . Indeed Tepper et al.(26) have shown that mutating Arg to Ala in Dictyostelium NDP kinase reduces the specific activity to 0.5% of normal. In this paper we describe a mutation at this site in Drosophila that also reduces NDP kinase specific activity. Thus the Kpn loop may be important for both the integrity of the hexamer and the active site. In order to understand the biochemical basis of prune/Killer of prune lethality, we recovered second site mutations which revert the prune/Killer of prune lethal interaction and analyzed the molecular basis of these mutations as well as the effects of these mutations on AWD/NDP kinase activity.


EXPERIMENTAL PROCEDURES

Recombinant AWD

awd cDNA was amplified from plasmid pc600(26) using primers 020 (CCCGGGCATATGGCGGCTAACAAGGAGAGG) and 009 (TATAGGATCCTATTACTATTCGTAGATCCAGTCCTT). The amplified fragment was subcloned into pET3C expression vector (28) as a NdeI/BamHI fragment to produce pAS28. The insert of pAS28 was sequenced before use and was found to be free of errors.

Purification of AWD Protein

BL21 cells containing expression plasmid pAS28 were cultured in LB media until an A of 0.4 was attained. The cultures were then induced with 0.5 mM isopropyl-1-thio-beta-D-galactopyranoside for 2 h. The cells were pelleted, resuspended at 4 °C in 1/100 original culture volume of HMED (10 mM HEPES, pH 7.4, 2 mM MgCl(2), 1 mM EDTA, 1 mM dithiothreitol, and 0.25 mM phenylmethylsulfonyl fluoride), and lysozyme and sonicated. Cell debris and unlysed cells were removed by centrifugation at 4 °C for 30 min at 8,000 times g. Ammonium sulfate was added to the supernatant to a final concentration of 40%, and the solution was stirred at 4 °C for 1 h. Precipitated proteins were pelleted at 4 °C for 30 min at 8,000 times g. Ammonium sulfate was added to the supernatant to a final concentration of 90% and stirred overnight at 4 °C. Precipitated proteins were pelleted at 8,000 times g for 30 min at 4 °C. The pellet was resuspended in HMED, pH 6.9, and was dialyzed against HMED, pH 6.9, at 4 °C. The protein solution was adsorbed onto a blue Sepharose CL-6B column equilibrated with HMED, pH 6.9 (Bio-Rad Econo-Column, 1.5 times 30 cm), washed with 10 column volumes of HMED, and eluted with 5 mM ATP/HMED, pH 8. AWD protein was dialyzed against HMED and rebound and eluted from a fresh column as before. The final protein eluate was dialyzed extensively against HMED, 10% glycerol for bioassays and long term storage or against coupling buffer for use in antibody purification.

Assay of NDP Kinase Activity

Third instar larvae or adults were homogenized in 100 µl of cold 100 mM Tris-HCl buffer, pH 7.4, containing 10 mM MgCl(2) and 100 mM KCl. After centrifugation for 5 min in a microcentrifuge at 4 °C, the supernatant was removed, diluted in buffer, and assayed for activity. The reaction mixture contained MgCl(2) (10 mM), KCl (100 mM), NADH (0.4 mM), ATP (6 mM), TDP (0.7 mM), P-enolpyruvate (4 mM), pyruvate kinase (10 units), lactate dehydrogenase (10 units), and 10 µl of diluted extract in a final volume of 1 ml of Tris-HCl buffer (100 mM, pH 7.4). Nonmutant extracts were diluted 1:49; mutant extracts were not diluted. Activity was measured as loss of NADH absorbance at 340 nm. A unit of activity is defined as the amount required to convert 1 µmol of NADH to NAD in 1 min at room temperature. Protein concentrations in the extracts were measured using the Bradford protein assay (Bio-Rad) with BSA as standard.

Preparation of Affinity Column

5 g of cyanogen bromide-Sepharose 4B affinity resin (Sigma) was washed sequentially with 1000 ml of 1 mM HCl then 150 ml of coupling buffer (100 mM NaHCO(2), 0.5 M NaCl, pH 8.8) on a sintered glass filter. The resin was poured into a Bio-Rad Econo-Column (1.5 times 30 cm) and agitated in a solution of 3.48 mg/ml purified recombinant AWD protein in coupling buffer for 4 h. The bound resin was washed with coupling buffer on a sintered glass filter and the resin was then resuspended in 1 M ethanolamine, pH 8, and agitated while in the column for 1 h. Ethanolamine was removed from the resin using a sintered glass filter. The resin was then resuspended in 200 ml of acetate buffer (0.1 M sodium acetate, 1 M NaCl, pH 4.0), loaded back into the column, then washed sequentially with 100 ml of acetate buffer, 100 ml of borate buffer (0.1 M borate, 1 M NaCl, pH 8.0), and finally 100 ml of phosphate buffer (10 mM NaPO(4), 0.3 M NaCl, pH 6.8).

Purification of High Affinity Antibody

Anti-AWD antibodies were produced in rabbits injected with purified, bacterially expressed AWD. High and low affinity antibodies against recombinant AWD protein were purified essentially as described by Kellogg and Alberts (29) for anti-tubulin antibodies. The AWD affinity resin was equilibrated with TBS buffer (20 mM Tris, 0.5 M NaCl, pH 7.5). 3 ml of polyclonal rabbit serum was diluted 1:3 with TBS, loaded onto the column, and then washed with TBS at a flow rate of 1 ml/min until A = 0. A low affinity antibody population was eluted from the column in 50 mM HEPES, 1.4 M MgCl(2), 10% glycerol, pH 7.6, and then dialyzed against phosphate-buffered saline, 50% glycerol. The column was washed with TBS, and a high affinity antibody population was eluted from the column with 0.5% acetic acid, 0.15 M NaCl. The eluted antibodies were immediately neutralized with 1 M NaHPO(4), stabilized with BSA, and stored in 0.05% sodium azide.

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting

Extracts of larvae were prepared in 5 mM Tris buffer, pH 7.5, containing 20% glycerol and 3% SDS. Protein concentrations were assayed using the DC protein assay system (Bio-Rad) and were adjusted to 0.4 mg/ml. 10 µl of each sample was boiled with 10 µl of 5 mM Tris, pH 7.5, buffer containing 20% glycerol, 3% SDS, and 85 mM beta-mercaptoethanol. 15 µl of each sample was run on a 15% polyacrylamide gel, electroblotted onto Immobilon-P (Millipore), blocked with 5% Carnation dried milk, 0.05% Tween 20 in phosphate-buffered saline, and incubated with high affinity polyclonal anti-AWD antibodies. Binding of goat anti-rabbit horseradish peroxidase-labeled secondary antibody (Amersham Corp.) was detected by enhanced chemiluminescence (Amersham). Molecular weight standards were low range prestained SDS-PAGE standard (Bio-Rad).

Agarose Isoelectric Focusing and Immunoblotting

Extracts of larvae or adults were prepared in 10 mM Tris, 10 mM KCl, 1 mM MgCl(2), pH 7.4. Protein concentrations were assayed using the Bradford protein assay (Bio-Rad) with BSA standard. The extracts were run on IsoGel agarose slabs (FMC BioProducts) pH range 3-10 using IsoGel pI markers (FMC BioProducts). The proteins were transferred by press-blot transfer to Immobilon-P. The blots were blocked with 5% Carnation dried milk, 0.05% Tween 20 in phosphate-buffered saline, and incubated with high affinity polyclonal anti-AWD antibodies. Binding of the goat anti-rabbit horseradish peroxidase-labeled secondary antibody (Amersham) to primary antibody was detected by enhanced chemiluminescence (Amersham).

Incubation of Purified Protein with Nucleotides

Purified recombinant AWD was dialyzed extensively against HMED buffer to remove ATP, diluted to 0.2 µg/µl, and incubated with various concentrations of dCTP or ADP for 5 min at room temperature. The protein in each reaction was resolved by isoelectric focusing in an IsoGel agarose gel. The AWD protein was detected by immunoblotting. To examine phosphate release from the AWD enzyme intermediate, aliquots of purified AWD protein (0.2 µg/µl) were incubated with 0.01 mM dCTP for 5 min at room temperature, then various amounts of ADP were added to each tube for an additional 5 min at room temperature. Each reaction mixture was fractionated by isoelectric focusing. The AWD protein was detected by immunoblotting using high affinity polyclonal anti-AWD antibody.

Gel Overlay Assay

Proteins in larval extracts were fractionated by isoelectric focusing in agarose gels and transferred by press-blot transfer to Immobilon-P filters. The filters were overlaid with 5 ml of 0.5% agarose that contained the coupled enzyme reaction mixture for detecting NDP kinase described by Lascu et al.(30) except that the substrate for detecting horseradish peroxidase was 5 mg/ml of 3.3`-diaminobenzidine tetrahydrochloride.

Generation of Prune/Killer of Prune Lethal Larvae

Females homozygous for yellow and prune mutations on the X chromosome were crossed to males homozygous for awd on the third chromosome. Since the prune gene is located on the X chromosome, all the male larvae resulting from this cross are yellow prune/Y; awd/awd (designated prune/Killer of prune) and are distinguishable from their sibling yellow prune/yellowprune; awd/awd females by the yellow phenotypic marker which is also on the X chromosome. The awd gene is located on the third chromosome and only one copy of awd is required for lethality of prune males.

Mutagenesis

Male flies (0-24 h old) homozygous for awd were fed overnight with 12.5 mM ethyl methanesulfonate suspended in 1% sucrose. The mutagenized males were mated to females homozygous for prune and ebony mutations. Male progeny of this cross represent putative revertants. These males were mated to females heterozygous for an awd null mutation to test for allelism.

Southern Blotting

Restriction digests of genomic DNA prepared from 20 larvae or adults were electrophoresed on 1% agarose gels run in Tris acetate buffer. After electrophoresis, the gels were processed in 1.5 M NaCl, 0.5 M NaOH, and then 3 M NaCl, 0.5 M Tris, pH 8. The DNA was transferred onto HyBond-N filters (Amersham) with 20 times SSC. The filters were UV-irradiated for 4 min, prehybridized in 0.5% SDS, 6 times SSC, 5 times Denhardt's solution, and 250 µg/ml denatured salmon sperm DNA, and hybridized at 65 °C overnight in the same solution with denatured, radiolabeled probe added. Filters were washed in 2 times SSC, 0.1% SDS at 65 °C for 5 min., then 2 times SSC, 0.1% SDS at 65 °C for 1 h. Hybridization was detected by autoradiography.

Amplification and Sequencing of awd Mutants

Genomic DNA was prepared from third instar larvae homozygous for the induced mutation or from larvae transheterozygous for the induced mutation and awd, which is a large (over 5.5 kb) deletion of the awd region(20) . The DNA was amplified using primer 010 (GACTGAATTCAGATCTTGAACCGCTACGCC), which binds 790 bp upstream from the 5` end of awd cDNA, and primer 019 (GAGCAACAGAGCTGTGG), which binds 770 bp downstream from the 3` end of awd cDNA. The amplified mutant fragments were subcloned into pCRII cloning vector (Invitrogen) for subsequent DNA sequence analysis. Sequencing was performed on double-stranded plasmid DNA using standard protocols and U. S. Biochemical Corp. Sequenase II sequencing kit.


RESULTS

Purification of Recombinant AWD/NDP Kinase

AWD/NDP kinase was expressed in bacteria and was purified according to the protocol of Lascu et al.(21) with slight modifications. On SDS-PAGE the purified protein migrates as a single band of 17 kDa (Fig. 1) as expected from its predicted molecular weight deduced from the nucleotide sequence of an awd cDNA (20) and in agreement with the results of Lascu et al.(21) for AWD/NDP kinase purified from adult flies.


Figure 1: Purification of recombinant AWD protein. The AWD protein was purified as described under ``Experimental Procedures'' with samples of each purification step reserved for analysis on polyacrylamide gels. Proteins were fractionated on 15% polyacrylamide-SDS gels and detected by Coomassie Blue staining.



Specificity of an Antibody Raised against AWD/NDP Kinase

In order to detect wild-type and mutant forms of AWD/NDP kinase on immunoblots, rabbit polyclonal antiserum was generated and purified as described. The antibodies used in these experiments were the high affinity antibodies eluted from an AWD affinity column and are referred to here as HA1 (see ``Experimental Procedures''). The specificity of HA1 immunoreactivity on immunoblots was tested on extracts of wild-type and awd mutant larvae fractionated by SDS-PAGE (Fig. 2A) or by agarose isoelectric focusing, IEF (Fig. 2B). The mutant used, awd, has a 788-bp deletion that removes the entire protein coding region of awd(31) and serves as a negative control. On immunoblots of SDS-PAGE gels, HA1 recognizes a single band at 17 kDa in extracts of wild-type larvae, and does not recognize any proteins in awd mutant larvae. On immunoblots of IEF gels, the HA1 antibody recognizes a major AWD isoform with a pI of 7.4 and minor isoforms with pI values of 7.7 and 7.1 in extracts of wild-type larvae and does not recognize any proteins in awd mutant larval extracts. This antibody preparation is therefore very specific for the AWD/NDP kinase protein.


Figure 2: Specificity of HA1 antibody. Extracts of larvae were fractionated by SDS-PAGE (A) or IEF-agarose (B). Proteins were transferred to filters and immunoblotted with HA1 antibody. AWD/NDP kinase is detected in wild-type larvae (+) but not in mutant larvae(-). Mutant larvae were homozygous for awd which is missing the entire awd coding region.



Multiple Banding Pattern on IEF Immunoblots Is Due to Phosphorylation

Lascu et al.(21) observed minor bands of protein when they fractionated purified AWD/NDP kinase on IEF gels, and they suggested that these minor protein bands represented either contaminants or minor isoforms of the enzyme. The NDP kinase reaction occurs via a ping-pong mechanism utilizing a phosphorylated enzyme intermediate, and the phosphorylation occurs on a histidine residue(15) . We hypothesized that the multiple bands detected on isoelectric focusing gels represent phosphorylated intermediates of the hexameric AWD/NDP kinase, the most acidic isoform representing a hexamer with all six subunits phosphorylated at the active site. In a test of this hypothesis, we incubated purified recombinant AWD/NDP kinase with increasing concentrations of dCTP as a phosphate donor, fractionated the protein by IEF, and immunoblotted with HA1. With increasing concentration of dCTP, the number of bands increased to a total of seven major isoelectric forms (Fig. 3). The pI of the most acidic major isoform was 7.1. The observed shift to more acidic isoforms with increasing substrate concentrations can be attributable to either nucleotide binding or phosphorylation of the protein. We do not believe that the acidic modification is due to nucleotide binding, because it does not occur in the presence of nucleoside diphosphates (data not shown), and because NDP kinase incubated with alpha-P-labeled nucleoside triphosphates (NTPs) hardly becomes labeled at all compared with the intense labeling that occurs when incubated with -P-labeled NTPs ((11) ; our data not shown). Therefore, we interpret the shift to more acidic AWD isoforms which occurs in the presence of dCTP to be due to phosphorylation of the protein. Since no protein kinase was added to the reaction, and since we have used a substrate, dCTP, which is unlikely to be used by a protein kinase, the most likely site of phosphorylation is the active site histidine. According to this interpretation such phosphorylated forms represent enzymatic intermediates. If so, then subsequent incubation with a nucleoside diphosphate should cause transfer of phosphate from the enzyme and loss of acidic forms of the enzyme(32) . That result is indeed what is observed (Fig. 4).


Figure 3: Acidic modification of AWD/NDP kinase by incubation with dCTP. Purified AWD/NDP kinase was incubated with dCTP at indicated molar concentrations, fractionated by isoelectric focusing in an agarose gel, and immunoblotted with HA1 antibody. Arrows indicate pI of each species calculated from the position of markers.




Figure 4: Reversal of dCTP acidic modification of AWD/NDP kinase by incubation with ADP. Purified AWD/NDP kinase was incubated with 10M dCTP and then incubated with ADP at indicated molar concentrations. The protein was fractionated by isoelectric focusing in an agarose gel and immunoblotted with HA1 antibody.



The Different Isoelectric Forms of AWD/NDP Kinase Are Enzymatically Active

We have modified a colorimetric assay for NDP kinase (30) to detect enzyme activity in gel overlays. We tested extracts of wild-type larvae fractionated by IEF for immunoreactivity and for enzyme activity using this modified colorimetric assay. All the IEF bands which are immunoreactive are also enzymatically active (Fig. 5). If we assume that the amount of protein in each band detected by immunoblotting is proportional to the mass of AWD protein in each band, then the amount of enzyme activity detected in the overlay assay suggests that the more acidic isoforms have a higher specific activity.


Figure 5: Comparison of immunoreactivity and enzyme activity of the different isoforms. Extracts of wild-type larvae were fractionated by isoelectric focusing in an agarose gel, transferred to a filter, and either immunoblotted with HA1 antibody (left) or overlaid with a reaction mixture to detect NDP kinase activity colorimetrically (right).



The Prune Mutation Does Not Reduce the Enzymatic Activity of Wild-type or Mutant AWD/NDP Kinase

Lascu et al.(21) showed that the awd mutant form of AWD/NDP kinase is enzymatically active. Our results are similar. We find that the specific activity in crude extracts of awd mutant larvae is about one-third the level of wild-type specific activity (Table 1). This level of activity is more than enough for normal development (31) and, indeed, awd homozygous larvae develop normally. In prune mutant larvae, the specific activity is similar to that of normal larvae (Table 1), and prune larvae also develop normally. In prune/Killer of prune lethal larvae, the NDP kinase specific activity is also similar to that of wild-type larvae (Table 1). Thus, the lethality of prune/Killer of prune larvae is not due to an inadequate level of NDP kinase activity.



The awdMutation Does Not Cause Accumulation of KPN/NDP Kinase Monomers

Wild-type larval extracts fractionated by IEF have multiple NDP kinase isoforms which we interpret as NDP kinase hexamers with varying numbers of phosphorylated active site histidines. Extracts of viable prune mutant larvae, viable awd mutant larvae, and lethal prune/Killer of prune mutant larvae have identical patterns of multiple AWD isoforms (Fig. 6). The relative intensities of the bands from mutant larvae are comparable with the relative intensities of the bands from wild-type larvae. If the awd mutation caused accumulation of KPN/NDP kinase monomers, then the relative abundance of IEF isoforms would be altered in viable awd mutant larvae and in lethal prune/Killer of prune mutant larvae. An increase in the amount of protein in the most basic pI 7.7 band would be expected if unphosphorylated monomers were accumulating, whereas an increase in the intensity of the most acidic pI 6.4 band would be expected if phosphorylated monomers were accumulating. The relative abundance of IEF isoforms are similar in extracts from wild-type, prune, awd, and prune/Killer of prune larvae; therefore, accumulation of KPN/NDP kinase monomers does not occur in awd larvae.


Figure 6: The distributions of different isoforms is not altered in prune, awd, or prune/Killer of prune larvae. Larval extracts of the indicated genotype were assayed for protein concentration. Equal amounts of protein were fractionated by isoelectric focusing in an agarose gel, transferred to a filter, and immunoblotted with HA1 antibody. Lane 1, yellow prune/Y viable male larvae; lane 2, yellowprune/Y; awd/awd viable male larvae; lane 3, yellow prune/yellowprune; awd/awd viable female larvae; lane 4, yellow prune/Y; awd/awd lethal male larvae; lane 5, Canton-S wild-type male larvae; lane 6, homozygous null mutant yellow/Y; awd/awd lethal male larvae; lane 7, purified recombinant AWD/NDP kinase; lane 8, Canton-S wild-type male larvae.



Recovery of Ethyl methanesulfonate-induced Revertants of awd

We previously reported the recovery of -ray induced revertants of awd(20) . Most of those revertants were chromosome rearrangements such as deletions which removed part or all of the awd gene. In order to investigate the mechanism of prune/Killer of prune lethality, we repeated the mutant screen using Ethyl methanesulfonate as the mutagen. This modified screen is more likely to produce point mutations in protein coding regions in the awd gene or in another gene which could revert the prune/Killer of prune lethality. We report 12 new revertants which were recovered at a frequency of 1/15,000 chromosomes tested. All of them are loss of function alleles of awd and all of them have significantly less NDP kinase activity than wild type. Most have a level of activity similar to that of the deletion allele awd (Table 2).



Biochemical Analysis of awdRevertants

Southern blots of genomic DNA extracted from revertant homozygous larvae were probed with an awd genomic region to look for rearrangements within the awd gene. The wild-type awd gene is contained within a 2.2-kb PstI fragment. Revertants awd and awd are missing the entire awd gene as well as flanking DNA (Table 2). Revertant awd has a small deletion within the awd coding region (Table 2). This truncated fragment was amplified by PCR and its nucleotide sequence was determined. awd has a deletion of 788 bp, which removes the entire coding region of awd without disturbing either flanking gene. It serves as our standard for a complete null allele.

The remaining nine revertants have a normal-size PstI fragment. The initiator methionine in two of these revertants, awd and awd, is changed to lysine and valine, respectively, and these two revertants fail to accumulate AWD/KPN protein. The remaining seven revertants contain single point mutations which result in single amino acid changes ( Table 2and Fig. 7) in addition to the Kpn mutation. In awd, the arginine at position 89 is changed to cysteine. In awd, arginine at position 106 is changed to cysteine. In awd the glutamic acid at position 130 is changed to lysine. In both awd and awd, the aspartic acid residue at position 15 is changed to asparagine. In awd, alanine at position 127 is changed to threonine; and in awd, aspartic acid at position 122 is changed to asparagine (Table 2, Fig. 7).


Figure 7: Amino acid replacements in awd revertants. Nucleotide changes and names of mutations are indicated above the wild-type sequence; amino acid replacements are indicated below the sequence. The revertants awd and awdhave the identical nucleotide change but are of independent origin.



Extracts from control larvae and each of these nine point mutant revertants were fractionated by IEF and immunoblotted with HA1. All of the revertants and controls were assayed as hemizygotes; they had one revertant copy of the awd gene and one copy of awd. We used awd as a positive control, since all of the point mutant revertants were induced in an awd mutant background. Three major isoforms were detected in awd hemizygotes with approximate isoelectric points of pH 7.4, 7.1, and 6.8 (Fig. 8). We used the awd coding region deletion allele awd as a negative control. A very faint band is detected in awd homozygotes with an approximate isoelectric point of pH 7.4 (Fig. 8). We interpret this faint band to be residual maternally supplied NDP kinase(31) . The same faint band is also detected in another negative control, awd, and in all of the revertants. The mutant, awd, has a normal awd coding region but a mutation in the regulatory region which severely reduces awd transcription. (^2)Neither awd nor awd revertant larvae accumulate AWD protein (Fig. 8), which is expected from the loss of an initiator methionine mutation. The level of NDP kinase specific activity in these extracts is not greater than in awd larvae (Table 2). The AWD protein in awd revertant larvae (Arg Cys, Table 2) and in awd revertant larvae (Arg Cys, Table 2) gives rise to a single band on IEF agarose gels which is indicative of nonphosphorylated subunits (Fig. 8). The level of NDP kinase activity in extracts from either of these revertants is as low as the specific activity of the null awd mutant. The isoelectric point of awd and awd AWD protein (6.1) is more acidic than unphosphorylated wild-type AWD due to the replacement of Arg with Cys. The AWD protein in the awd revertant (Glu Lys, Table 2) produces multiple bands on IEF agarose gels, which is indicative of phosphorylated subunits. However, this mutant has a NDP kinase specific activity as low as that of the awd null mutant. A more basic isoelectric point is expected for the AWD protein in awd revertant larvae because of the replacement of an acidic residue with a basic residue, and indeed in some preparations we detect a pI 8.0 isoform which is more basic than the most basic isoform in control awd larvae. The AWD protein in awd and awd revertant larvae (Asp Asn, Table 2) gives rise to only a single band on IEF agarose gels which is indicative of nonphosphorylated subunits (Fig. 8). The AWD protein in these revertants is expected to be more basic due to the loss of an acidic Asp residue; however, these revertant AWD proteins have a more acidic isoelectric point than wild type (Fig. 8). Extracts from awd and awd have an NDP kinase specific activity as low as that of the null awd mutant. The AWD protein in awd revertant larvae (Ala Thr, Table 2) has multiply phosphorylated hexamers and has 20% of the enzyme activity of awd. Indeed we detect a major band with an isoelectric point of 7.4, which is the same as the most basic band in awd extracts. The AWD protein in awd revertant larvae (Asp Asn, Table 2) is expected to be more basic due to the loss of an acidic Asp residue; however, this revertant produces primarily a single more acidic isoform. Extracts from awd revertant larvae have 20% of the awd-specific enzyme activity. For some of the revertants that should synthesize normal amounts of protein, such as awd, awd, awd, awd, and awd, reaction with the antibody is more intense or less intense than with the positive control (Fig. 8). This is either because the double mutant protein has different stability than the KPN protein or because the HA1 antibody preparation binds to the double mutant protein differently than it does to the KPN protein.


Figure 8: Revertants alter the accumulation or isoelectric point of AWD/NDP kinase. Each lane represents a hemizygous mutant larval extract. For each mutation indicated, larvae transheterozygous for that mutation and awd were selected by their larval yellow phenotype. Mutant larval extracts were assayed for protein concentration and equal amounts of total protein were fractionated by agarose IEF and immunoblotted with HA1 antibody. Samples of the positive control awd/awd and the negative control awd/awdwere fractionated on both of two gels.




DISCUSSION

There Is Only One Drosophila Gene, awd, That Encodes Cytosolic NDP Kinase

In humans there are two genes that encode cytosolic NDP kinase, nm23-H1 and nm23-H2(2, 3) . NDP kinase purified from human erythrocytes is a mixed hexamer composed of subunits derived from each gene(14) . We have found no evidence for a second Drosophila gene encoding a cytosolic NDP kinase. NDP kinase purified from Drosophila adults is a hexamer composed of only one subunit form(21) , and the isoelectric point of NDP kinase purified from flies (21) is similar to the isoelectric point of purified recombinant AWD/NDP kinase (Fig. 2B). Nevertheless, in awd null mutants there is 2-5% of residual NDP kinase activity(31) . We propose that this residual activity is either maternally derived AWD/NDP kinase or derived from mitochondrial NDP kinase that is the product of a separate gene yet to be identified in Drosophila. It has been known for many years that mitochondria have NDP kinase activity(33) . Recently in Dictyostelium, a mitochondrial NDP kinase derived from a separate, highly diverged gene has been isolated. The coding regions for Dictyostelium mitochondrial and soluble NDP kinase are only 57% identical(34) . For comparison, the sequences of Dictyostelium cytosolic NDP kinase and Drosophila AWD/NDP kinase are 58% identical. So Dictyostelium cytosolic NDP kinase is as similar to Drosophila AWD/NDP kinase as it is to Dictyostelium mitochondrial NDP kinase.

Multiple Bands Observed on IEF Gels Can Be Accounted for as Enzymatic Intermediates

Isoelectric focusing has revealed the occurrence of multiple species of NDP kinases in human erythrocytes (35) . These multiple forms were interpreted as related isoforms of the enzyme(1) . When it was demonstrated that human cytosolic NDP kinase is encoded by two different genes (nm23-H1 and nm23-H2), it seemed plausible that these multiple isoelectric species represented hexamers with different combinations of NM23-H1/NDP kinase A and NM23-H2/NDP kinase B subunits. However in Drosophila, only one gene encodes cytosolic NDP kinase, and yet multiple bands are also seen on isoelectric focusing gels (Fig. 2B, Fig. 5, and Fig. 6). We have presented evidence that all of these species in Drosophila can be accounted for as phosphorylated enzymatic intermediates. It is significant that recombinant protein incubated with high concentrations of NTP produces seven isoelectric species. Since Drosophila NDP kinase is a hexamer(13, 21) , we propose that the seven isoelectric species represent hexamers in which zero to six of the subunits are phosphorylated. Bominaar et al.(32) have investigated the autophosphorylation of Dictyostelium NDP kinase and concluded that essentially all of the phosphorylated forms of Dictyostelium NDP kinase represent enzymatic intermediates. This interpretation is identical to ours for purified Drosophila NDP kinase. Munoz-Dorado et al.(36) and MacDonald et al.(37) on the other hand have presented evidence for autophosphorylation on Ser residues of Myxococcus NDP kinase and human NM23-H1/NDP kinase A respectively. If autophosphorylation occurs on Ser residues of purified recombinant Drosophila AWD/NDP kinase, it occurs at a level below our limits of detection.

The isoelectric point (8.3) of the most basic isoform of purified, recombinant AWD/NDP kinase (Fig. 3) is more basic than the isoelectric point (7.7) of the most basic isoform in extracts of fly larvae (Fig. 2B, Fig. 5, and Fig. 6). One possible explanation for this difference in isoelectric point between AWD/NDP kinase produced in bacteria and AWD/NDP kinase produced in flies is that the enzyme produced in flies is stably phosphorylated on a residue other than the active site histidine leading to a more acidic isoelectric point than predicted by the amino acid composition. However, another possible explanation for this difference is that the alpha-amino group of the amino-terminal residue of AWD/NDP kinase produced in flies is modified which could also lead to a more acidic isoelectric point than that predicted by the amino acid composition. This latter possibility is made credible by the finding that the alpha-amino group of the amino-terminal residue of rat NDP kinase-alpha is indeed blocked(4) .

awdRevertants Affect Substrate Binding and/or Catalysis

The data presented here show that the dominant lethality caused by KPN mutant subunits in a prune background can be reverted either by preventing the accumulation of KPN subunits or by reducing the enzyme activity of the KPN subunits. In addition to the large revertant deletions previously reported, we have found five new revertants which prevent the accumulation of mutant subunits. Three of the five, awd, awd, and awd have deletions of the awd coding region; two of the five, awd and awd, have replacements of the initiator methionine, Met^1 Lys and Met^1 Val, respectively. None of these revertants accumulates KPN subunits, and as hemizygotes all five of these mutations cause an awd null phenotype indistinguishable from awd homozygotes(31) .

Seven additional revertants accumulate mutant subunits with amino acid substitutions. The mutant subunits in five of these seven awd, awd, awd, awd, and awd have no NDP kinase activity and as hemizygotes in a prune background, all five of these mutations also cause an awd null phenotype indistinguishable from awd homozygotes. The mutant subunits in the other two of the seven revertants that accumulate KPN protein, awd and awd, have approximately 20% of the enzyme activity of awd. Both awd and awd heterozygotes are viable in a prune mutant background. So at a minimum 80% of KPN/NDP kinase activity must be eliminated to escape lethality in the absence of prune gene function. In the presence of prune gene function awd hemizygotes are viable and have 28% of wild-type NDP kinase activity; awd hemizygotes produce some viable adults and have 6% of wild-type NDP kinase activity. Thus the minimal amount of NDP kinase activity required for viability is between 6 and 28% of the wild-type activity. We arrived at a similar conclusion in a different experiment using a promoter-awd cDNA transgene(31) .

The seven revertants we recovered that accumulate enzymatically inactive or less active AWD subunits affect six different conserved residues. Two of those residues, Arg and Arg hydrogen-bond to the beta phosphate of bound nucleotides(11, 19, 38) . Glu positions the active site His(11, 19, 38) . Tepper et al.(26) have shown by site-directed mutation of the Dictyostelium cytosolic NDP kinase that alteration of any of these three residues dramatically reduces enzyme activity, in complete agreement with our results. The Arg Cys and Arg Cys mutant proteins (from awd and awd, respectively) have no activity and do not accumulate phosphorylated subunits, which suggests that the amino acid substitutions in these mutant proteins severely affect the initial binding of NTP substrate. The isoelectric points of these mutant forms migrate according to the predicted value, which is another indication that the mutants are not phosphorylated. The Glu Lys mutant protein in the awd revertant does accumulate phosphorylated forms of the protein so binding of NTP substrates must occur. Since the turnover rate for the NDP kinase phosphorylation reaction is much slower than the turnover rate for the dephosphorylation reaction(1) , and since phosphorylated forms of the Glu Lys mutant protein do accumulate, we expected to observe a reduced enzymatic activity for this mutant. Yet instead of a reduced level of enzymatic activity, we detect no significant activity for this mutant. Our interpretation of these observations is that the active site histidine is capable of becoming phosphorylated in such a mutant yet at a much reduced rate, because the active site histidine is no longer positioned properly by Glu. A reduction of the dephosphorylation rate must also occur to completely eliminate activity of the mutant enzyme. We propose three mechanisms by which the dephosphorylation reaction rate could be reduced. 1) Suboptimal positioning of the phosphorylated histidine by the mutant residue might affect the rate of phosphate transfer to bound NDP. 2) Migration of the phosphate residue from the 1 to the 3 position of histidine, which is the more stable form, might occur in the mutant protein and this isomeric form might have a reduced rate of phosphate transfer. 3-Phosphohistidine residues have been identified previously (1) , but the affect of 3-phosphohistidine on the catalytic activity of the enzyme has not been thoroughly examined. 3) The fact that we observe negligible NDP kinase specific activity with this mutant rather than simply a reduction in NDP kinase specific activity is probably also a reflection of the charge interference by the Lys substitution at position 130. The mutant Lys residue might stabilize the phosphorylated intermediate, preventing its transfer to NDP. The other mutations that we have isolated, Asp Asn, Ala Thr, and Asp Asn, also reduce enzyme activity, and we propose that they also disrupt substrate binding and/or catalysis. From the crystal structure data it is known that Lys hydrogen bonds to the sugar residue of nucleotides(11, 19, 38) . We suggest that replacement of Asp by Asn in awd and awd disrupts this interaction and causes the loss of enzyme activity (Table 2). The fact that we do not observe multiple phosphorylated forms with the two Asp Asn revertants suggests that this is indeed the case and that the Lys hydrogen bond is very important for nucleotide binding. The Asp Asn mutant proteins accumulate a more acidic form rather than the more basic form expected from the loss of an acidic Asp residue. One possible explanation for this is that the acidic form accumulating is actually the completely phosphorylated hexamer. If this explanation is correct, then the Asp Asn substitution preferentially affects NDP binding and subsequent dephosphorylation of the enzyme intermediate. The replacement of Ala by Thr in awd results in an 80% reduction of enzyme activity. We suggest that since Ala and Glu residues are on the same face of the alpha4 alpha-helix, the Ala change might disrupt the interaction of Glu and His. The fact that we see multiply phosphorylated forms with the Ala Thr protein and with the Glu Lys mutation is in agreement with this suggestion. Furthermore, since the Ala Thr substitution is much less drastic a change than Glu Lys, one would expect a less severe consequence from an Ala Thr substitution. This expectation is met by a less reduced specific activity, a less severe phenotype, and not as much accumulation of the phosphorylated Ala Thr enzyme intermediates as in the Glu Lys revertant. In addition, no change in the isoelectric banding pattern is predicted from this amino acid substitution, and no difference in pI from wild-type is observed. Finally, the Asp Asn replacement in awd causes an 80% reduction of enzyme activity and accumulation of an acidic form. We suggest that this change interferes with the transfer of phosphate from His to nucleoside diphosphates leading to the accumulation of hexamers in which all of the subunits are phosphorylated, analogous to the awd Glu Lys revertant. An alteration of the side chain interaction between Ser with the active site His might occur because of the Asp Asn substitution. This could explain why an acidic isoelectric form accumulates from the Asp Asn revertant (Fig. 8) despite the loss of an acidic residue (Fig. 7). The accumulation of hexamers in which all of the subunits are phosphorylated is never observed in AWD/NDP kinase extracted from wild-type or awd larvae and is only observed in purified AWD/NDP kinase that is incubated with extremely high concentrations of nucleoside triphosphates (Fig. 3).

Three different hypotheses have been presented to account for the conditional dominant lethality of the awd mutation in a prune background. The first was presented by Sturtevant (25) who discovered the phenomenon. He proposed that a substrate of the protein encoded by the prune gene accumulates in prune mutants and can be converted into a toxic substance by the awd gene product but not by its wild-type counterpart. The second hypothesis was presented by Teng et al.(22) and elaborated by Ruggieri and McCormick (39) and Hackstein(40) . They proposed that in awd mutants, NDP kinase is hyperactive and deregulated. The transcription of awd is certainly not deregulated; it is identical to the transcription of wild-type awd(20) . If it is meant that KPN/NDP kinase is hyperactive on normal substrates, then this hypothesis is not supported by fact that NDP kinase specific activity is slightly less in awd mutant larvae (Table 2). However, if by hyperactive it is meant that in awd mutants, KPN/NDP kinase has altered substrate specificity then this hypothesis becomes identical to Sturtevant's hypothesis. The third hypothesis was presented by Lascu et al.(21) and modified by Chiadmi et al.(13) . They hypothesized that the neomorphic function of the mutant KPN protein results from deleterious effects of monomeric (or dimeric) NDP kinase that is proposed to accumulate in awd larvae. This hypothesis was based on their observations that KPN/NDP kinase purified from awd homozygotes binds to normal substrates only slightly differently than wild-type protein, but that this mutant form of AWD/NDP kinase is more thermolabile and fails to renature after denaturation in urea. Their hypothesis is consistent with the idea based on the x-ray structure that the Kpn loop is involved in subunit interactions(12) . However, there is no evidence of accumulation of monomeric NDP kinase either in viable awd homozygous larvae or in prune/Killer of prune lethal larvae (this paper).

Our hypothesis for the conditional dominant lethality of awd (Fig. 9) is similar to the one originally proposed by Sturtevant in 1956 and is based on the data presented here implying that revertants we have recovered affect the enzymatic function of KPN/NDP kinase. We propose that the mechanism of prune/Killer of prune lethality depends on altered substrate specificity of AWD/NDP kinase subunits carrying the Pro Ser amino acid substitution caused by the awd mutation. As with other NDP kinases(1) , wild-type AWD/NDP kinase has a broad range of substrate specificity. The Kpn loop defined by Dumas et al.(12) positions the nucleotide binding cleft of the enzyme and indeed moves upon substrate binding(11) . Our model is that the Pro Ser substitution within the Kpn loop alters the nucleotide binding cleft, allowing an even broader range of substrate specificity. In a prune background no accumulation of potentially harmful abnormal substrates occurs. However, according to our hypothesis, in a loss of function prune mutant, a substrate accumulates that can be phosphorylated by KPN mutant subunits of NDP kinase but cannot be acted upon by wild-type AWD/NDP kinase. The inability of wild-type NDP kinase to act upon this hypothetical substrate explains why loss of prune function is normally not lethal. The dark eye color of prune mutant adults is due to loss of pteridine eye pigments which are derived from GTP. Hackstein (40) has reported altered metabolism of guanosine injected into prune mutants. One of these metabolites could represent the hypothetical substrate we propose accumulates in prune mutants. According to this interpretation, prune/Killer of prune lethality is due to harmful effects caused by the phosphorylation of a molecule that accumulates in prune mutants. Ultimate proof of this hypothesis awaits determination of the enzymatic activity of the prune gene product and the identities of its normal substrates.


Figure 9: Model of lethal interaction of prune and awd. A, wild type. The product of prune converts substrate X to Y; the product of awd converts NDPs to NTPs. B, awd mutant. The product of prune converts substrate X to Y; the product of awd converts NDPs to NTPs. C, prune mutant. Substrate X accumulates, because it cannot be converted to Y; the product of awd converts NDPs to NTPs. D, prune;awd/awd double mutant. Substrate X accumulates because it cannot be converted to Y; the product of awd converts NDPs to NTPs and phosphorylates substrate X converting it to a toxic substance.




FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM33959 and American Cancer Society Grant DB-91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 410-516-7285; Fax: 410-516-5213; bio\_cals{at}jhuvms.hcf.jhu.edu.

(^1)
The abbreviations used are: NDP, nucleoside diphosphate; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; kb, kilobase pair(s); bp, base pair(s); IEF, isoelectric focusing.

(^2)
L. Timmons and A. Shearn, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Michel Veron and Joel Janin for discussing their work prior to publication and for commenting upon this manuscript. We also thank Marsha Tharakan and Gloria Lim for performing NDP kinase assays and Jennifer Martin for critical review of the manuscript.


REFERENCES

  1. Parks, R., and Agarwal, R. (1973) in The Enzymes (Boyer, P. D., ed) pp. 307-344, Academic Press, New York
  2. Steeg, P. S., Bevilacqua, G., Kopper, L., Thorgeirsson, U. P., Talmadge, J. E., Liotta L. A., and Sobel, M. E. (1988) J. Natl. Cancer Inst. 80,200-204 [Abstract]
  3. Stahl, J. A., Leone, A., Rosengard, A. M., Porter, L., King, C. R., and Steeg, P. S. (1991) Cancer Res. 51,445-449 [Abstract]
  4. Kimura, N., Shimada, N., Nomura, K., and Watanabe, K. (1990) J. Biol. Chem. 265,15744-15749 [Abstract/Free Full Text]
  5. Shimada, N., Ishikawa, N., Munakata, Y., Toda, T., Watanabe, K., and Kimura, N. (1993) J. Biol. Chem 268,2583-2589 [Abstract/Free Full Text]
  6. Steeg, P. S., Bevilacqua, G., Pozzatti, R., Liotta, L. A., and Sobel, M. E. (1988) Cancer Res. 48,6550-6554 [Abstract]
  7. Urano, T., Takamiya, K., Furukawa, K., and Shiku, H. (1992) FEBS Lett. 309,358-362 [CrossRef][Medline] [Order article via Infotrieve]
  8. Nomura, T., Yatsunami, K., Honda, A., Sugimoto, Y., Fukui, T., Zhang, J., Yamamoto, J., and Ichikawa, A. (1992) Arch. Biochem. Biophys. 297,42-45 [Medline] [Order article via Infotrieve]
  9. Zhang, J., Nomura, T., Yatsunami, K., Honda, A., Sugimoto, Y., Moriwake, T., Yamamoto, J., Ohta, M., Fukui, T., and Ichikawa, A. (1993) Biochim. Biophys. Acta 1171,304-306 [Medline] [Order article via Infotrieve]
  10. Biggs, J., Hersperger, E., Steeg, P. S., Liotta, L. A., and Shearn, A. (1990) Cell 63,933-940 [Medline] [Order article via Infotrieve]
  11. Williams, R. L., Oren, D. A., Munoz-Dorado, J., Inouye, S., Inouye, M., and Arnold, E. (1993) J. Mol. Biol. 234,1230-1247 [CrossRef][Medline] [Order article via Infotrieve]
  12. Dumas, C., Lascu, I., Morera, S., Glaser, P., Fourme, R., Wallet, V., Lacombe, M. L., Veron, M., and Janin, J. (1992) EMBO J. 11,3203-3208 [Abstract]
  13. Chiadmi, M., Morera, S., Lascu, I., Dumas, C., LeBras, G., Vernon, M., and Janin, J. (1993) Structure 1,283-293 [Medline] [Order article via Infotrieve]
  14. Gilles, A. M., Presecan, E., Vonica, A., and Lascu, I. (1991) J. Biol. Chem. 266,8784-8789 [Abstract/Free Full Text]
  15. Edlund, B., Rask, L., Olsson, P., Wålinder, O., Zetterqvist, Õ., and Engström, L. (1969) Eur. J. Biochem. 9,451-455 [Medline] [Order article via Infotrieve]
  16. Saraste, M., Sibbald, P. R., and Wittinghofer, A. (1991) Trends Biochem. Sci. 15,430-434
  17. Schulz, G. E. (1992) Curr. Opin. Struct. Biol. 2,61-67 [CrossRef]
  18. Rossmann, M. G., Liljas, A., Branden, C. I., and Banaszak, L. J. (1975) Enzymes 11,61-102
  19. Morera, S., Lascu, I., Dumas, C., LeBras, G., Briozzo, P., Veron, M., and Janin, J. (1994) Biochemistry 33,459-467 [Medline] [Order article via Infotrieve]
  20. Biggs, J., Tripoulas, N., Hersperger, E., Dearolf, C., and Shearn, A. (1988) Genes & Dev. 2,1333-1343
  21. Lascu, I., Chaffotte, A., Limbourg-Bouchon, B., and Veron, M. (1992) J. Biol. Chem. 267,12775-12781 [Abstract/Free Full Text]
  22. Teng, D. H., Engele, C. M., and Venkatesh, T. R. (1991) Nature 353,437-440 [CrossRef][Medline] [Order article via Infotrieve]
  23. Frolov, M. V., Zverlov, V. V., and Alatortsev, V. E. (1994) Mol. & Gen. Genet. 242,478-483
  24. Hadorn, E., and Mitchell, H. K. (1951) Proc. Natl. Acad. Sci. U. S. A. 37,650-665
  25. Sturtevant, A. H. (1956) Genetics 41,118-123 [Free Full Text]
  26. Tepper, A. D., Dammann, H., Bominaar, A. A., and Veron, M. (1994) J. Biol. Chem. 269,32175-32180 [Abstract/Free Full Text]
  27. Dearolf, C. R., Tripoulas, N., Biggs, J., and Shearn, A. (1988) Dev. Biol. 129,169-178 [Medline] [Order article via Infotrieve]
  28. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185,60-89 [Medline] [Order article via Infotrieve]
  29. Kellogg, D. R., and Alberts, B. M. (1992) Mol. Biol. Cell 3,1-11 [Abstract]
  30. Lascu, I., LeBlay, K., Lacombe, M. L., Presecan, E., and Veron, M. (1993) Anal. Biochem. 209,6-8 [CrossRef][Medline] [Order article via Infotrieve]
  31. Timmons, L., Hersperger, E., Woodhouse, E., Xu, J., Liu, L. Z., and Shearn, A. (1993) Dev. Biol. 158,364-379 [CrossRef][Medline] [Order article via Infotrieve]
  32. Bominaar, A. A., Tepper, A. D., and Veron, M. (1994) FEBS Lett. 353,5-8 [CrossRef][Medline] [Order article via Infotrieve]
  33. Pedersen, P. L. (1968) J. Biol. Chem. 243,4305-4311 [Abstract/Free Full Text]
  34. Troll, H., Winckler, T., Lascu, I., Muller, N., Saurin, W., Veron, M., and Mutzel, R. (1993) J. Biol. Chem. 268,25469-25475 [Abstract/Free Full Text]
  35. Cheng, Y. C., Agarwal, R. P., and Parks, R. E., Jr. (1971) Biochemistry 10,2139-2143 [Medline] [Order article via Infotrieve]
  36. Munoz-Dorado, J., Almaula, N., Inouye, S., and Inouye, M. (1993) J. Bacteriol. 175,1176-1181 [Abstract]
  37. MacDonald, N. J., De la Rosa, A., Benedict, M. A., Freije, J. M., Krutsch, H., and Steeg, P. S. (1993) J. Biol. Chem. 268,25780-25789 [Abstract/Free Full Text]
  38. Cherfils, J., Moréra, S., Lascu, I., Veron, M., and Janin, J. (1994) Biochemistry 33,9062-9069 [Medline] [Order article via Infotrieve]
  39. Ruggieri, R., and McCormick, F. (1991) Nature 353,390-391 [Medline] [Order article via Infotrieve]
  40. Hackstein, J. H. P. (1992) Eur. J. Cell Bio. 58,429-444 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.