Text of The F ATP synthetase /J-subunit: a major yeast novobiocin ...Novobiocin affects DNA metabolism in...
The F1 ATP synthetase /J-subunit: a major yeast novobiocin binding protein
JOHN R. JENKINS, MICHAEL J. POCKLINGTON and ELISHA ORR
Department of Genetics, Adrian Building, University of Leicester, University Road, Leicester LEI 7RH, England
Novobiocin affects DNA metabolism in both pro-karyotes and eukaryotes, resulting in cell death. Inprokaryotes, the drug is a specific inhibitor of DNAgyrase, a type II topoisomerase that can be purifiedon a novobiocin-Sepharose column. The yeast typeII topoisomerase is neither the biochemical, nor thegenetic target of the antibiotic. We have purified themajor yeast novobiocin binding proteins and ident-ified one of them as the /J-subunit of the yeast mito-
chondrial Fi ATP synthetase, a protein highly con-served throughout evolution. The inactivation of thisprotein might explain the toxic effects of novobiocinon higher eukaryotic cells.
Coumarin antibiotics such as novobiocin have frequentlybeen used in the study of DNA metabolism (Orr et al. 1979;Fairweather et al. 1980; Gellert, 1981; Wang, 1985; Vos-berg, 1985). In prokaryotes, novobiocin has been identifiedas a specific inhibitor of DNA gyrase, a type II topoisomer-ase that introduces negative supercoils into relaxed, co-valently closed, double-stranded DNA molecules (Gellertet al. 1976). It has been suggested that the drug inhibitsbacterial gyrases by competing with ATP for the nucleo-tide binding site (Mizuuchi et al. 1978). Intriguingly, noATP binding domain has yet been identified either bio-chemically in the bacterial enzyme, or in its respectiveDNA sequence. Novobiocin has been reported to inhibitthe activity of some purified eukaryotic type II topoisomer-ases, though at concentrations 1000 times higher thanthose required to inhibit DNA gyrases. DNA sequenceanalysis of the prokaryotic and the eukaryotic enzymesreveals sequence homologies. For these reasons, it hasgenerally been presumed that the drug binds to homolo-gous sites on both the DNA gyrase and the eukaryoticenzymes.
Our recent data unambiguously demonstrate that yeasttype II topoisomerase is not the main target of novobiocin.Unlike bacterial gyrases, the enzyme does not bind to theantibiotic in vitro and the terminal phenotype of yeast top2its) mutants is different from the arrest phenotype of drug-sensitive yeast strains treated with novobiocin. Further-more, novobiocin-resistant mutants are not top2 mutants(Pocklington et al. 1990a). These conclusions are supportedby evidence obtained from other eukaryotic systems. BHKcell-lines resistant to novobiocin (Ishida et al. 1987)showed no alteration in topoisomerase II activity, and typeII topoisomerases from mouse cell lines could not beidentified among proteins eluted from a novobiocin-Sepharose column (Colwill and Sheinin, 1983).
Mattern and Scudiero, 1981; Mattern et al. 1982; Clarksonand Mitchell, 1983), transcription (Aller and Baserga,1986), condensation of chromosomes (Han et al. 1985;Villeponteau et al. 1984), the structure of mitochondriaand ATP synthesis (Downes et al. 1985; Dowries andJohnson, 1988). If the antibiotic does not inhibit theactivity of type II topoisomerase, as we have proposed,then the precise mechanism by which it exerts its effectremains unclear. Downes et al. (1985) demonstrated thatnovobiocin leads to the alteration of mitochondrial struc-tures in cultured cells. They further proposed that thetoxic effects of the antibiotic are caused by its interferencewith the ATP pool in the cell.
The affinity of bacterial gyrases to novobiocin has beenwell documented, as has its use in the purification of theenzyme (Staudenbauer and Orr, 1981; Orr and Stauden-bauer, 1982). We have applied a similar method in purify-ing yeast proteins with high affinity to novobiocin. In thispaper, we describe the purification and characterization ofone of these proteins, which has been identified as theyeast /3-subunit of the mitochondrial Fi ATP synthetase.The inactivation of this protein could probably affectprocesses involved in cellular growth. However, our re-sults obtained with yeast p° mutants, treated with novo-biocin, suggest though that the toxic effects of the anti-biotic in yeast might be mediated through its interferencewith other proteins rather than with the mitochondrialATP-synthesizing enzyme alone.
Materials and methods
StrainsBladder carcinoma cells, T24, grown on RPMI 5% foetal calfserum were a gift from N. Royle, and C0S8 and SW3T3 cellsgrown on DMEM+10% new-born calf serum were a gift from P.Jackson.
Saccharomyces cerevisiae, 842-1, 842 and ff 842 strains (Wattset al. 1987), were routinely grown on YPD (1 % yeast extract, 2 %bactopeptone and 2% glucose), ff strains were obtained by
growing the parent cells on YPD+0.5/igml l ethidium bromidefor 24 h. p° colonies were identified by their inability to grow onYPG (1 % yeast extract, 2 % bactopeptone and 2 % glycerol) and bystaining with DAPI, which stains the wild-type mitochondria!DNA.
Preparation of novobiocin-SepharoseThe novobiocin was coupled to Sepharose following the procedureof Studenbauer and Orr (1981). A 5 g sample of Epoxy-activatedSepharose CL6B (Pharmacia) was swollen in 500 ml distilledwater for 60 min at room temperature and washed on a scinteredglass filter with 500 ml of 0.3 M sodium carbonate, pH 9.5 (bufferA). The gel was then mixed with a solution of 500 mg novobiocin(Sigma) in 15 ml buffer A and gently shaken for 16 h at 37 °C.Excess Epoxy groups were blocked by the addition of ethanol-amine (final concentration 1 M) and shaking was continued foranother 4h at 37 °C. The product was then washed sequentiallywith 500 ml each of 0.5 M NaCl in buffer A, distilled water, 0.5 MNaCl in O.lM-sodium acetate, pH4.0, and distilled water. Carewas taken to protect the novobiocin from strong light at all times.
300 mA for 17 min, using a Western blotter Clear Ideas. Thetransferred proteins were visualized with Ponceau S (BDH). Non-specific binding sites were blocked by incubating the filter with3% BSA in 10 mM Tris-HCl, 150 mM NaCl (TBS), at 4°C over-night. The filters were washed twice with TBS and incubated witha serum raised against the p50 (1:500 dilution in TBS containing1% BSA) at 37 °C for 2h. The filter was washed four times withTBS and incubated with goat anti-rabbit IgG, washed four timeswith TBS and incubated with rabbit PAP (DAKO) for 60 min atroom temperature. After four washes with TBS, the complex wasdetected by a colour reaction as follows: 30 ml of TBS was added to10ml of methanol containing 30 mg 4-chloro-l-napthol and 30ftlof H2O2 (added last). The membrane was incubated in the mixture
Fractionation of cell extractYeast cells were grown in YPD medium to mid to late logarithmic(fermentative) phase, harvested by centrifugation and the pelletfrozen at -20°C. The cells were broken using a precooled (-20°C)Hughes press. The resulting cell lysate was made to 5 mM MgCl2and treated with deoxribonuclease I for 15 min at 4°C. An equalvolume of 2 M KC1 was added to the mixture, which was centri-fuged at 185 000g at 4°C for 60 min. The clear fraction of thesupernatant was carefully removed, diluted with buffer A con-taining no KC1 to a salt concentration of 0.1 M, and loaded on anovobiocin-Sepharose column. The column was washed success-ively with 1 M KC1, 2 M KC1 and 5 M urea in buffer B (25 mMHepes, pH 8.0, 1 mM dithiothreitol, 1 mM EDTA, 10 % (v/v) ethyl-ene glycol and 50 mM KC1) at 4°C.
Rat liver extract was kindly donated by Paul Jackson. Cyto-plasmic and nuclear extracts of T24 were prepared by homogeniz-ation followed by osmotic shock, and kindly provided by NicolaRoyle.
Amino acid sequencingThe /3-subunit of the Fi ATP synthetase was run on a 10%SDS-polyacrylamide electrophoresis gel and electroblotted ontopolyvinylidine difluoride (PVDF) membrane (Matsudaria, 1987)using transfer buffer made of 50 mM glycine, 50 mM Tris-HCl,pHIO, and stained with Coomassie Blue R-250. The stained bandwas excised and loaded onto an Applied Biosystem 470A gas-phase sequencer.
Western blotsFollowing SDS-PAGE, proteins were electroblotted onto nitrocel-lulose membranes as described by Towbin et al. (1982), or at markers (Mrx 10 ).
• 14Fig. 1. Coomassie staining of SDS-PAGE gel (10 %) of proteinseluted from novobiocin-Sepharose column. Yeast proteinsprepared from the Amyol strain 842-1 were applied to thecolumn and eluted with buffer B containing KC1 or urea. Lane,1, crude extract; lane 2, wash-through (100 mM KC1); lane 3, 1 MKC1; lane 4 , 2 M KC1; lane 5 , 5 M urea; lane 6, molecular weight
2 3 4 5
" I V6 7
Fig. 2. Immunoblot of cell extracts probedwith the anti-yeast /3-ATP synthetaseantibodies. Lane 1, pre-immune serum; lane 2,yeast 5 M urea fraction from the novobiocin-Sepharose column; lane 3, crude wild-typeyeast extract; lane 4, crude extract of ff cells;lane 5, E. coli proteins eluted with 5 M ureafrom the novobiocin-Sepharose column; lane6, crude wild-type yeast extract; lane 7, totalcell extract from chicken liver; lane 8,cytoplasmic extract from a bladder carcinomacell line; lane 9, nuclear extract from abladder carcinoma cell line.
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F|-ATP synthetase /S-subunit
(1) 5. cerevisiae
(2) 5. cerevisiae
Ifl 20 30 40 50
Fig. 3. Amino acid comparison of the fi-ATP synthetase peptide from a bovine source of protein (Runswick and Walker, 1983); (1) Astranslated from the yeast DNA sequence (Takeda et al. 1985); (2) the yeast N-terminal amino acid sequence determined in thisstudy. The numbers (10-50) for the amino acids are based on the translation data from Takeda et al. (1985).
by shaking at room temperature until the colour development wassatisfactory (generally 1-5 min), and washed with water.
Anti-50K (K=103 Mr) protein antibodiesThe proteins eluted from the novobiocin-Sepharose column with5 M urea were separated on SDS-PAGE and the region containingthe 50K protein was sliced out. It was mixed with equal volumesof sterile water and incomplete Freund's adjuvant, emulsified bysonicatdon and injected into rabbits to raise antibodies.
The anti-50K antibodies were affinity purified by incubatingthe rabbit serum with the 50K protein blotted onto a nitrocell-ulose strip. The latter was washed four times with TBS. Boundantibodies were eluted by shaking the filter strip with pre-cooled0.2 M glycine-HCl, pH2.2, at 4°C for 5 min. 1% BSA (final) wasadded to the eluent, which was neutralized with 2 M Tris-baseprior to dialysis against TBS containing 10 mM sodium azide.
Fixation of cellsT24, C0S8 and SW3T3 cells were grown on coverslips in Petridishes, washed twice in TBS and then fixed in TBS+3.7%formaldehyde at room temperature for 10 min. The cells werewashed once with TBS and once with sterile distilled water,covered with acetone (-20°C) and placed in a -18°C freezer for5 min. The acetone was removed and the cells were allowed to airdry before the primary IgG fraction was added.
S. cerevisiae cells were fixed with 3.7% formaldehyde asdescribed by Kilmartin and Adams (1984). The fixative waswashed thoroughly with TBS and the cells resuspended in 1.2 Msorbitol containing 50 mM Tris-HCl, pH 7.5, and 10 mM /S-mercap-toethanol. The cell walls were digested with 50/jgml"1 (final)zymolyase at 30°C until the cells turned phase-dark (approxi-mately 30 min). The cells were washed three times with TBS,resuspended in TBS containing 0.2% Triton X-100 (Sigma) andleft for 10 min at room temperature. They were then washed threetimes with TBS and stored in 1.2M sorbitol at -20°C for indirectimmunofluorescence microscopy.
Staining proceduresThe DNA of fixed cells was stained using Hoechst 33342 (Sigma)at a dilution of lO/igml"1 in 0.1 % p-phenylenediamine (Sigma),90% glycerol and 10 mM Tris-HCl, pH7.5.
For indirect immunofluorescence microscopy, fixed cells wereincubated with the affinity-purified antibodies at a dilution of1:20 in TBS at 37 °C for 2h, washed three times in TBS andincubated with FTTC-conjugated goat anti-rabbit IgG (TAGO) at adilution of 1:250.
The vital stain DASPMI was used essentially as described byBereiter-Hahn (1976). The cells were washed several times with50 mM Tris-HCl, pH8.0, containing 150 mM NaCl and incubated,in the dark, with the dye dissolved in the same buffer at aconcentration of 10~8 to 10~6moll~1 at room temperature for30-60 min.
All microscopy was carried out using the Zeiss Axiophotphotomicroscope as described by Williamson and Fennel (1975).
Purification of high-affinity novobiocin binding proteinsFive major novobiocin binding proteins have been purifiedfrom crude yeast extract by affinity chromatography on anovobiocin-Sepharose column. Bound proteins wereeluted from the column by washing with 1.0 M KC1, 2.0 MKC1 and 5 M urea. Fractions containing proteins wereanalysed by SDS-PAGE. The 2 M salt buffer eluted twoproteins from the column: one is a 200K protein that hasbeen identified and characterized as a yeast myosin heavychain (Watts et al. 1985,1987). The other is a 52K protein,the product of a gene conferring novobiocin resistance toyeast (Pocklington et al. 19906). Extracts used in thisstudy were rountinely prepared from strain 842-1, aAmyol strain (Watts et al. 1987).
Owing to their greater affinity for novobiocin, elution of
Fig. 4. Indirect immunofluorescence microscopy of yeast cellsstained with: A, DAPI and; B, affinity-purified anti-yeast /3-ATPsynthetase antibodies (secondary FITC). X1000.
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Fig. 5. Indirect immunofluorescence microscopy showing similar staining patterns of Cos8 cells stained with: A, affinity-purifiedanti-50K antibodies (secondary FITC); and B, DASPMI (x900).
the other three proteins of approximately 200K, 50K and20K from the column required denaturing conditions with5 M urea (see Fig. 1). The 50K protein, p50, could specifi-cally be washed off the column with 10 mM ATP. Theprotein was further purified by SDS-PAGE and electro-eluted from the gels to raise antibodies. Western blotanalysis of total yeast proteins and the novobiocin bindingproteins indicates that the antibodies against the p50recognise a single band with the same mobility in bothfractions (Fig. 2).
N-terminal amino acid sequence of the 50K novobiocinbinding proteinTwenty residues from the N-terminal end of the 50Knovobiocin binding protein were determined without any
ambiguities. These were used to scan the Swissprot pro-tein data base. A perfect match was found between thissequence and that of the S. cerevisiae Fi ATP synthetase /S-subunit, the deduced relative molecular mass of which is54575 (Takeda et al. 1985). Details of the two sequencesare presented in Fig. 3. The yeast sequence in Fig. 3 is thatof the mature mitochondrial protein, indicating that thecleavage point of the yeast mitochondrial delivery peptideis at residue 31. This result is in agreement with thatdescribed for the homologous protein from a bovine mito-chondrial source (Runswick and Walker, 1983).
AntiSOK immunological studies of yeast cellsThe fixing of yeast cells was achieved by using eitheracid-methanol or formaldehyde. Although acid-methanol
Fig. 6. Indirectimmunofluorescence microscopy ofyeast cells stained with: A and C,DAPI; and B and D, affinity-purified anti-yeast /J-ATPsynthetase antibodies: A and B,untreated; C and D, treated with1 mgml"1 novobiocin for 2 h(X1400).
678 J. R. Jenkins et al.
fixation is a faster process, it can lead to the distortion ofinternal structures such as the microtubules and themicrofilaments. Yeast cells fixed with acid-methanol andstained with the affinity-purified anti-50K IgG indeedshow distorted structures when compared with cells fixedwith formaldehyde. S. cerevisiae cells fixed with formal-dehyde and stained with the affinity-purified anti-50K IgGand DAPI (4',6-diamidino-2-phenyl-indole) are shown inFig. 4. The results clearly indicate that the localization ofthe antibodies coincides with the staining of the mitochon-drial DNA by DAPI.
p° yeast strains lack mitochondrial DNA; thus theirmitochondria are energetically inactive and are unable togrow on non-fermentative media. Consequently, they can-not be visualized with the vital stain DASPMI (dimethyl-aminostyrylmethyl-pyridiniumiodine), which is specificfor the energetically active mitochondrial membrane (Ber-eiter-Hahn, 1976). We therefore stained p° strains with theaffinity-purified anti-50K IgG to see whether the proteincan be transported into the energetically inactive mito-chondria. The results with the p° strains were similar tothose obtained with the wild-type strain, indicating thatthe ATP synthetase subunit enters the mitochondria. AWestern blot analysis of total cell proteins from both thewild-type and the p° strains demonstrates that the p50proteins from both strains have the same apparent rela-tive molecular mass (Fig- 2). This result is compatiblewith our conclusion that the /J-subunit of the p° f\ ATPsynthetase is processed by the removal of the mitochon-drial delivery sequence.
Cross species homology of SOK proteinsMitochondrial proteins are known to be highly conservedstructures within eukaryotic cells. We consequently separ-ated, by SDS—PAGE, total chicken liver proteins andproteins isolated from the cytoplasm and the nuclei of abladder carcinoma cell line, T24. Nitrocellulose blots ofthese proteins were then probed with the anti-50K anti-bodies (Fig. 2). A single band with a similar mobility tothat of the yeast protein was detected in both the chickenliver proteins and the cytoplasmic extract from the T24cell line. Three mammalian cell lines, T24, COS8, SW3T3and one plant (tulip) tissue culture, were grown oncoverslips and used in indirect immunofluorescence mi-croscopy studies. Mitochondria from all the cell linestested stained positively. COS8 and SW3T3 cells stainedwith DASPMI and the affinity-purified anti-50K IgG areshown in Fig. 5 and Fig. 7A,B, respectively, indicatingthat both recognise mitochondria.
The Escherichia coli 5 M urea fractionIt has previously been reported that the B-subunit ofbacterial DNA gyrase is the major novobiocin-bindingprotein in bacteria (Staudenbauer and Orr, 1981). Pro-karyotes are known to contain a protein homologous to thep'-subunit of the yeast Fx ATP synthetase. Logically, thisprotein should have been identified among the bacterialnovobiocin binding proteins. Our results prompted us tore-examine the E. coli proteins that bind tightly to novo-biocin-Sep'harose. Fig. 2 shows a Western blot analysis ofthe E. coli proteins eluted from the column with 5 M ureaand probed with the yeast anti-ATP synthetase antibodies.It is clear that the antibodies cross-react with a polypep-tide of approximately the same relative molecular mass asthe yeast protein. Moreover, like the latter, the E. coliprotein can be eluted specifically from the novobiocin-Sepharose column with ATP.
Fig. 7. Indirect immunofluorescence microscopy of SW3T3 cells;A and B, untreated; C, treated with 1 mgml"1 novobiocin for2 h; A, stained with DASPMI; both B and C, stained with theanti-yeast /J-ATP synthetase antibodies (secondary FITC). X666.
The effect of novobiocin on mitochondriaWe have reported (Pocklington et al. 1990a) that drug-sensitive yeast cells treated with novobiocin have adistinctive morphology when examined microscopically.Fluorescence microscopy of these cells stained with DAPIreveals that after 2h in the presence of novobiocin themitochondrial DNA forms clusters of condensed particles.After a prolonged treatment with the antibiotic (3-6 h)
Fi ATPase synthetase fi-subunit 679
Fig. 8. Indirect immunofluorescence microscopy of T24 cells at various stages of mitosis, stained with: A, DAPI; and B, anti-yeast /S-ATP synthetase antibodies (secondary FTTC). x500.
these particles scatter throughout the cell. Fig. 6 under-lines our previous results obtained by DAPI staining. Itdemonstrates, using antibody staining, that the condensedparticles are not fragments of the nucleus, but are mito-chondria.
Mitochondrial DNA in mammalian cells cannot bevisualized properly with DAPI. We therefore used theantibodies and DASPMI to evaluate the effect of novobio-cin on these cells. The mitochondria in the latter appear tobe affected in a similar manner to that observed in yeast,namely they become condensed and distorted (Fig. 7).
In mammalian cells the mitochondria assemble aroundthe nucleus after the nuclear membrane has broken downand separate along with the two sets of the daughterchromosomes. Previous reports suggested that mitochon-dria and other organelles are attached to the microtubularcytoskeleton (Linden et al. 1989). Our results (Fig. 8)similarly indicate an attachment of the mitochondria tothe microtubular network. This type of extensive network,
however, is missing from S. cerevisiae. We have observedthat mitochondria enter the daughter bud well ahead ofthe nucleus (date not shown). Such orientated movementis presumably achieved by the activity of a cytoskeletalnetwork.
Sequence comparison of the GyrB and the ATPsynthetase fi-subunit proteinsA comparison of the B-subunit of the E. coli DNA gyrasewith the /3-subunit of the Fx ATP synthetase reveals verylittle homology at the amino acid level, apart from a fiveamino acid region: YQPTL. The /3-subunit of the ATPsynthetase was then compared with the B-subunit ofBacillus subtilis DNA gyrase. In this case even lesshomology at the amino acid level was observed. The twopossible matches with the YQPTL motif both required twomismatches: AQPPL and LQVTL. However, both P and Vare conservative substitutions for their counterparts. We
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think that this motif represents a possible novobiocinbinding domain.
It is evident from our results that the coumarin antibiotic,novobiocin, alters mitochondrial structure. We proposethat these structural effects are mediated through thebinding of the drug to the 0-subunit of the FX ATPsynthetase. Interference with the ATP-synthesizing en-zyme should be sufficient to arrest cellular growth, if ATPproduction is halted. Unlike other eukaryotes, however,yeast can grow on fermentative media without energeti-cally functioning mitochondria. In the latter case, novobio-cin still inhibits cellular growth, indicating that its poi-soning effects in yeast are exerted either via aninactivation of the mitochondrial membranes essential forfatty acid synthesis or via other components of the cell. Wehave purified four other major yeast novobiocin bindingproteins. Among these, we have characterized a 52Kprotein - the product of the wild-type allele of a geneconferring novobiocin resistance in yeast (Pocklington etal. 19906). Inactivation of the Nov protein is lethal andcould therefore account for the inhibitory effects of theantibiotic. An analogous situation can be observed inbacteria. Our results demonstrate that novobiocin binds tothe prokaryotic ATP synthetase /S-subunit. Nevertheless,mutants resistant to the drugs are defective in GyrB,which binds tightly to the antibiotic. It is possible that thebinding of novobiocin to the bacterial ATP synthetase doesnot entirely inactivate the enzyme. Alternatively, theaffinity of the antibiotic for the ATP synthetase subunitmight be lower than to the DNA gyrase, leading primarilyto the selection of DNA gyrase mutants. Finally, it ispossible that all mutants defective in the ATP synthetasesubunit are lethal.
Is there a primary protein sequence that constitutes anovobiocin binding domain? Such a site has not been foundin bacterial DNA gyrases. It has been speculated thatnovobiocin and ATP compete for the same binding domain,as yet unidentified. It is plausible that there is more thanone novobiocin binding domain in different proteins,explaining the poor homology between gyrases and the fi-subunit of the Fi ATP synthetase. However, it is temptingto speculate that the five conserved amino acids in both theE. coli DNA gyrase and the ATP synthetase subunits arean essential part of the novobiocin binding domain. InBacillus subtilis gyrase there are two groups of aminoacids each containing three of the five conserved aminoacids. Perhaps the three conserved amino acids are theessential part of the site, permitting a conservative substi-tution of the other two, with no significant loss of novobio-cin binding activity. The proposed novobiocin binding siteis well separated from the nucleotide binding site in the /S-subunite of the ATP synthetase. This might facilitate thebinding of ATP to its exposed binding site, leading toconformational changes in the protein and consequently toits elution from the novobiocin column.
We thank Nicola Royle and Paul Jackson for the cell lines usedin this study. We also thank Mathew Davieson for the amino acidsequencing service. This research was supported by the MRC(UK) grant no. G8301270 and the Cancer Research Campaign.
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