Plant Molecular Biology 18: 873-885, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.

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eDNA clones encoding Arabidopsis thaliana and Zea mays mitochondrial chaperonin HSP60 and gene expression during seed germination and heat shock Tottempudi K. Prasad and Cecil R. Stewart* Department of Botany, Iowa State University, Ames, IA 5001I, USA (* author for correspondence) Received 9 August 1991; accepted in revised form 25 November 1991

Key words: Arabidopsis thaliana, ATPase, cpn60, developmental regulation, molecular chaperones, Zea mays

Abstract

Mitochondria contain a nuclear-encoded heat shock protein, HSP60, which functions as a chaperonin in the post-translational assembly of multimeric proteins encoded by both nuclear and mitochondrial genes. We have isolated and sequenced full-length complementary DNAs coding for this mitochondrial chaperonin in Arabidopsis thaliana and Zea mays. Southern-blot analysis indicates the presence of a single hsp60 gene in the genome ofA. thaliana. There is a high degree of homology at the predicted amino acid levels (43 to 60~o) between plant HSP60s and their homologues in prokaryotes and other eukaryotes which indicates that these proteins must have similar evolutionarily conserved functions in all organisms. Northern- and western-blot analyses indicate that the expression of the hsp60 gene is developmentally regulated during seed germination. It is also heat-inducible. Developmental regulation of the (/~-subunit of F1-ATPase, an enzyme complex that is involved in the cyanide-sensitive mitochondrial electron transport system, indicates that imbibed embryos undergo rapid mitochondrial biogenesis through the early stages of germination. Based on the functional role of HSP60 in macromolecular assembly, these data collectively suggest that the presence of higher levels of HSP60 is necessary during active mitochondrial biogenesis, when the need for this protein is greatest in assisting the rapid assembly of the oligomeric protein structures.

Introduction

Molecular chaperones are a class of essential proteins whose function is to ensure the correct folding and assembly of other polypeptides into oligomeric structures of which the chaperones are

not a component [15,21]. Chaperonins are a sub-class of chaperones, to which belong the family of heat shock proteins (HSPs) with a molecular mass of 60000 Da that include GroEL in Escherichia coli, ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) subunit binding

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers Z 11546 and Z 11547.

874 protein (RBP or plastid Cpn60) in chloroplasts and HSP60 (mitochondrial Cpn60) in mitochondria. The GroEL or chaperonin-60 (Cpn60) and GroES or chaperonin-10 (Cpnl0)proteins are part of a functional complex in bacteriophage head protein assembly in E. coli [16]. Using in vivo and in vitro analyses, Goloubinoff et at. [17, 18] demonstrated that both Cpn60 and Cpnl0 are required for the assembly of prokaryotic Rubisco in a (Mg-ATP) hydrolysis-dependent manner. While the existence of a GroES homologue was also discovered in animal mitochondria [31], a similar GroES homologue has yet to be reported to exist in plants. Both nuclearencoded polypeptides [4, 6, 41] and organelle (mitochondria- and chloroplast)-encoded polypeptides [1, 44, 49] have been shown to be associated with chaperonins. While the functions of chaperonins in the assembly of other polypeptides into macromolecular structures are well documented [14, 15, 28, 47, 48], their own assembly from monomers was reported to depend on self-stimulation in a (Mg-ATP)-dependent manner [29]. The genes encoding chaperonins from several organisms have been cloned and the derived amino acid sequences show a high degree of conservation from prokaryotes to eukaryotes [8, 21, 23, 24, 34, 46]. Some of the chaperonins are known to be HSPs in both prokaryotes and eukaryotes. While all of the HSPs could possibly be chaperonins, not all the chaperonins are heatinducible. Members of the HSP70 family were shown to be required for protein translocation into mitochondria and endoplasmic reticulum [7, 10] and were also reported to be involved in macromolecular assembly along with HSP60 in mitochondria [25 ]. Consistent with the hypothesis proposed by Pelham [42, 43] concerning the function of HSPs, the HSP70 homologue in E. coli, the dnaK gene product, was demonstrated to contribute to cell growth not only by protecting some enzymes from heat denaturation but also by reactivating some others once they are unfolded or aggregated [53]. There are several reports on the accumulation of high and low-M r HSPs in plants [26, 32, 33, 39, 56, 57, 59]. How-

ever, except for HSP60 [44, 45] and ubiquitin [5], there is no direct evidence indicating their function. It is known that several HSPs are developmentally regulated in various plants and animals under normal temperatures, in addition to their induction by environmental stresses [2, 20, 56, 57]. Early events in seed germination have been used to define mechanisms by which mitochondrial biogenesis is regulated. Electron microscopic studies show that quiescent seed tissues contain poorly differentiated mitochondria but, upon imbibition, they become enlarged and develop complex inner membrane structures [37]. The development of functional mitochondria during imbibition was reported to be dependent on de novo protein synthesis in peanut cotyledons [37] and on the activation of preformed mitochondria in pea cotyledons [36]. It was reported in germinating maize embryos that the cyanide-sensitive mitochondrial electron transport system was required for embryo germination and this activity depended upon newly synthesized or assembled respiratory enzyme complexes [ 13 ]. In the present study, we report the isolation, sequencing, and characterization of full-length cDNAs encoding the mitochondrial assembly factor, HSP60, from A. thaIiana and Z. mays. Additional information is presented on the involvement of HSP60 in mitochondrial biogenesis during maize embryo germination.

Materials and methods Plant material

Seeds of Zea mays (cv. Black Mexican Sweet) were imbibed overnight in running water and then germinated in wet vermiculite in the dark at 25 ° C for 24 to 120 h. For heat shock treatments, 120 h old whole seedlings were incubated in 1~o sucrose plus 4 mM potassium phosphate, pH 6.0, at 37 °C for 3 h in a gyratory shaker. The nonheat treated seedling shoots were harvested at regular intervals and used for mitochondrial isolation or total R N A isolations. Arabidopsis

875

thaliana cv. Columbia seeds (a gift from Dr Martin Spalding, Iowa State University, Ames) were germinated on 1 To water agar plates for 24 to 120 h in the dark at 25 ° C. For heat shock treatments, whole 120 h old seedlings were incubated at 37 °C for 3 h. Entire seedlings were used for mitochondrial and total RNA isolations.

Mitochondrial and chloroplast protein extractions Mitochondria were isolated according to the method of Day and Hanson [9]. In brief, tissue was homogenized in a grinding buffer (0.4 M sucrose, 5 ~o insoluble PVP, 5 mM EGTA, 1.0 mg/ ml BSA, 50 mM TES, pH 7.6) and the homogenate was filtered through four layers of cheesecloth and centrifuged for 10min at 3000 x g. Mitochondria were further purified by centrifugation through a sucrose cushion. Chloroplasts from light-grown pea leaves were isolated according to the method of Blair and Ellis [3]. In brief, leaf tissue was homogenized in a grinding buffer (0.35 M sucrose, 25 mM HEPES pH 7.6, 2 mM EDTA, 2 mM sodium isoascorbate) and passed through 8 layers of cheesecloth. The homogenate was centrifuged at 2500 x g for 1 min. The chloroplast pellet was washed in homogenization buffer and recentrifuged at 2500 x g for 1 min. Both organelle proteins were extracted by boiling in Laemmli sample buffer for one minute.

Purification of Hsp6O from maize for polyclonal antibody production and N-terminal protein sequence analysis Purified mitochondria were lysed with 1/20th volume of 10~o sodium deoxycholate plus 20~o Triton X-100 followed by gentle vortexing. The lysate was fractionated on a 15 to 30~o sucrose gradient [35] and the HSP60-enriched fraction was resolved on two-dimensional PAGE [40], isoelectric focusing (IEF) in the first dimension followed by SDS-PAGE in the second dimension. The proteins from the gel were transferred

to Immobilon-PVDF membrane (Millipore) and stained with Ponceau S. The HSP60 protein spot on the membrane was marked with a pencil. The membrane was destained in distilled water, marked spots were cut out and the protein was eluted into the non-ionic detergent solution. The protein was precipitated with cold acetone and the pellet was air-dried before resuspending into phosphate buffer. A total of approximately 150 #g of purified HSP60 was used to raise polyclonal antibodies in a rabbit by standard protocols. Of the protein l#g was used for N-terminal sequence determination on an Applied Biosystems Model 470 sequenator and the sequence of first 36 residues were identified.

Western blotting Mitochondrial proteins were resolved on SDSPAGE using 11 ~ gels. The proteins were transferred to nitrocellulose membranes and probed either with anti-HSP60 polyclonal antiserum (1:5000 dilution) raised against purified maize HSP60 or with anti-F1-ATPase (/3-subunit) monoclonal antibodies (1:100 dilution) raised against maize/%subunit of F1-ATPase (a gift from Dr T. Elthon, University of Nebraska, Lincoln). The secondary antibody was alkaline phosphataseconjugate (Sigma) and the color detection was done using nitroblue tetrazolium/bromo-chloroindolyl phosphate [22]. Proteins for westerns were loaded on to the gels in equal amounts as determined by spectrophotometric analysis followed by visual observations of stained gels (data not shown). Changes in the levels of HSP60 or /%subunit of F ~-ATP ase immunoreactive protein s on western between treatments was done by the visual estimations.

Screening of cDNA expression libraries with antiHSP60 antiserum Z. mays (cv. Black Mexican Sweet) and A. thaliana cDNA libraries constructed in expression vectors pUC 13 (a gift from Dr J. Hunsperger,

876 University of Minnesota, St. Paul) and 2gtll (Clonetech Labs.), respectively, were screened with anti-HSP60 antiserum raised against purified maize HSP60. Several positive c D N A clones were isolated from both the libraries. The largest cDNA insert fragments of maize (2.14 kb) and A. thaliana (2.0 kb) clones were subcloned into 'phagemid' vectors, pUC 118 or pUC 119 [55]. Plasmid DNA was isolated by the alkaline lysis method and purified by CsCl-ethidium bromide gradient centrifugation [50].

DNA sequencing After preliminary restriction mapping analysis of the maize cDNA clone, suitable restriction fragments were subcloned into pUC 118 or pUC 119. Since no restriction sites for most of the common restriction enzymes were found in the A. thaliana cDNA clone, subfragments for sequence analysis were obtained by digestion with Bal 31 nuclease [ 50] and recloned into pUC 118 or pUC 119 [ 55 ]. In addition, oligonucleotides (21-mers)were synthesized according to sequence information obtained and used directly as primers for further sequencing, wherever necessary. The sequence analysis was performed by the dideoxynucleotide chain-termination method [ 51] and the sequences of both D N A strands were determined. Computer analysis of nucleotide and deduced amino acid sequences and their alignments were performed using the programs of the University of Wisconsin Genetics Computer Group [ 11]. Sequence data from other organisms were obtained from the GenBank (update release 68.0, June 1991).

tion buffer (50~o formamide, 5 x SSC, 1 x Denhardt's solution, 150/~g/ml denatured herring sperm DNA, 20/~g/ml carrier RNA (yeastsoluble), 0.2~o SDS) and then hybridized overnight at 42 °C in the same buffer with 7~o dextran sulfate. The probe was prepared with gelpurified A. thaliana cDNA insert labeled with 32p-dCTP (3000 Ci/mmol, Amersham) by random hexamer priming. The filter was washed at room temperature in 2 x S S C , 0.1~o SDS for 15min with two changes and once with 0.1 x SSC, 0.1~o SDS for 30 min at 50 °C. Lowstringency hybridizations were not performed with an assumption that HSP60 probe might show non-specific hybridization with genes encoding plastid Cpn60.

Northern-blot analysis Total RNAs were isolated from control or heatshocked germinating seedlings of maize and A. thaliana according to the method of Haffner et al. [ 19]. Equal amounts of RNA were electrophoresed in 1~o formaldehyde agarose gels and transferred to nitrocellulose filters. Stained gels were observed to confirm that equal amounts of RNA were contained in each sample (data not shown). The filters were prehybridized, hybridized and washed as described for Southern-blot analysis. The respective gel-purified cDNA inserts were labeled with 32p-dCTP (3000 Ci/mmol, Amersham) by the random hexamer priming technique and used as probes. Changes in transcript levels were estimated visually. Results

Southern-blot analysis

Specificity of anti-Hsp60 antiserum raised against purified HSP60 from maize

Genomic DNA from A. thaliana was isolated according to the CTAB method [12]. DNA was digested with Hind III, Eco RI and Pst I restriction enzymes, resolved on 0.7~o agarose gels followed by transfer to nitrocellulose filters. Filters were prehybridized for 2 h at 42 °C in hybridiza-

Previous studies with anti-Hsp60 antiserum (raised against HSP58 from Tetrahymena thermophila) indicated no significant cross-reactivity with purified pea plastid Cpn60 even at relatively high concentrations [45]. Since the antiserum, in the present studies, was raised against

877 purified HSP60 from a plant source, it becomes further important to determine its specificity to mitochondrial HSP60, not to plastid Cpn60. To test this, mitochondria from etiolated maize seedlings and chloroplasts from pea leaves were isolated. The organelle proteins at various concentrations were resolved on SDS-PAGE. Identical gels were either stained with Coomassie blue or transferred to nitrocellulose for western analysis. The stained gel shows typical mitochondrial or chloroplast protein profiles (Fig. 1) which indicates that the organelle preparations were highly enriched for mitochondria and chloroplasts. Western blot analysis clearly shows that antiHSP60 specifically reacts with an approximately 62 kDa mitochondrial HSP60 but does not show any detectable cross-reactivity in chloroplast proteins even at 3-fold higher protein levels compared to mitochondrial proteins (Fig. 1).

Isolation and sequence analysis of cDNAs Maize and A. thaliana c D N A expression libraries were screened with anti-HSP60 antiserum and c D N A clones that code for Hsp60 were isolated. The c D N A inserts were subcloned and the complete D N A sequence was determined on both strands. The nucleotide sequences of cDNAs and the deduced amino acid sequences are presented in Fig. 2. Both maize and A. thaliana c D N A sequences contain single, long open reading frames of 1.731 kb which code for polypeptides of 577 amino acids in length each with a relative molecular mass of 6 1 3 1 2 D a and 61260 Da, respectively. These predicted values are close to the determined values of the mature proteins [45]. In both maize and A. thaliana, the termination codons after the 1.731 kb coding region were followed by additional termination codons in the same frame. At the 3' end of the sequence, there are polyadenylation consensus sequences 'AATAAA' 127 bp from the stop codon in A. thaliana and 186 bp from the stop codon in maize. The determined amino-terminal amino acid sequence of mature maize protein (36 residues) matches 100~o with that of the predicted amino-

Fig. 1. Specificity of anti-HSP60 antiserum (raised against purified maize HSP60) with HSP60 and/or plastid Cpn60 in maize mitochondrial and pea chloroplast proteins, respectively. Proteins from purified maize mitochondria (lanes 1 and5) and pea chloroplasts (lanes2-4 and6-8) were extracted as described in the Materials and methods section. The respective lanes contain 60 #g of mitochondrial proteins (lanes 1 and 5) and 60 #g (lanes 2 and 6), 120 #g (lanes 3 and 7) or 180 #g (lanes 4 and 8) of chloroplast proteins. The proteins were resolved on SDS-PAGE. Identical gels were either stained with coomassie blue (lanes 1-4) or transferred to nitrocellulose membrane and probed with anti-HSP60 antiserum (lanes 5-8). Molecular weight markers (M r in kDa) are indicated.

terminal amino acid sequence in maize and 93 ~o with that of the predicted amino-terminal amino acid sequence in A. thaliana, starting with the 35th residue in maize and the 32nd residue in A. thaliana as shown in Fig. 2 (amino acid sequence between closed arrows). Examination of the predicted sequences of the amino-terminus from the start codon reveals that they are rich in basic and hydroxylated residues, but deficient in acidic amino acid residues. While there is no homology at the nucleotide level in the 5' and 3' untranslated region, the coding regions show 76~o homology at the nucleotide level and 86~o at the predicted amino acid level between the maize and A. thaliana cDNAs (Fig. 2). The regions of homology, both at the nucleotide and amino acid levels, are distributed evenly throughout the sequence length. When codon usage was examined between maize and A. thaliana, the third base of

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T~ ~CGAC~C ~ ~A~ ~ A ~ il lili lillll I II I li II T~TGA~C ~C~TCCAT~T T ~ L

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GAGGCAT ~ T G G T GGT CATGGAC T A C T A G A T C C AC TAAAT TCT GT CTT TT GTT TT CAC CAATCAC CATCT T A A ~ ~ T A C ~ T ~ i lliillIilll I il llill i I I I i I i I II II ii G C G G C A T G G G T G G C A T G ......... CAT T A C T A A T G C A T C A C A C C C A C A T A C A C A TCAAT G C A G ~ T T T T T TTTTA G C G G T G A A G T T ~ ~CAC T~ ~ G

1933

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1998

i lill il i I I i i I I I i il GT CAGGC G G A T A C A GTT TT GAGTC TGC G G A C G T A G ~ A C C T C A C A A T A G T T C C A A A ~ T A G T G T G A G A A T ~ T A T CT GTATT CGATC G T T A C A T T T T T T G G C T G G G G T A A A A TC GGA TT GCC GTTCT G T ~ A A A A A A A A A A A A A T T T

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879 the synonymous codons was different in over 50 ~o of the codons.

Homology between plant HSP60s and their homologues

The deduced amino acid sequences of maize and A. thaliana HSP60s and their homologues in organisms ranging from bacteria to higher eukaryotic species are compared in Fig. 3 and Table 1. Since no homology exists in the presequences between plant HSP60s and their homologues, the homology was analyzed only for the mature parts of the polypeptides (starting with amino acid 35). With the introduction of only a few gaps, the maize and A. thaIiana HSP60s show high degrees of similarity to their homologues (Fig. 3 and Table 1). Notably, the partial amino acid sequences of plastid Cpn60 c~ and/%subunits in A. thaliana are quite different from HSP60 sequences in the same species with only 44-46~o homology. This observation supports our conclusion that we isolated mitochondrial Hsp60 cDNA, not the plastid form of Cpn60 c D N A (Table 1). The regions of homology are distributed evenly throughout the length of the polypeptides (Fig. 3). Interestingly, there is a highly conserved repeat of a GG-M motif at the carboxyl terminus of the plant HSP60s and their homologues except in plastid Cpn60.

Gene organization in A. thaliana To determine the structure and organization of the genes homologous to the HSP60 m R N A corresponding to the c D N A clone, total A. thaliana

genomic DNA was digested with restriction enzymes Hind III, Pst I and Eco RI and hybridized to a labeled A. thaliana cDNA clone insert on Southern filters. Figure 4 shows that, in each digest, there is only one restriction fragment that hybridized with the probe, suggesting that there is a single HSP60 gene in the A. thaliana genome.

Developmental regulation of HSP60 and atp2 (/?subunit of F1-A TPase) gene expression during seed germination Northern blots of total RNA hybridized with labeled maize orA. thaliana HSP60 and maize atp2 cDNA, inserts are presented in Fig. 5. The dry seeds of A. thaliana and dry seed embryos of maize contained levels of HSP60 transcripts higher than that contained in seedlings 120 h after germination (Fig. 5A, B). The maize dry seed embryos also contained some transcripts of the atp2 gene (Fig. 5C). During imbibition and germination, all the investigated transcripts accumulated 15-fold or more within 24 h and then declined to 5- and 3-fold by 48 and 120 h, respectively, in both A. thaliana and maize. The mitochondrial proteins on western blots were reacted with anti-HSP60 polyclonal antibodies or anti-/%subunit of F1-ATPase monoclonal antibodies (Fig. 5). Like the m R N A levels, immunoreactive HSP60 levels in dry seeds and embryos of A. thaliana and maize were 2-fold higher than the levels in the seedlings at 120 h of germination (Fig. 5A, B). In contrast, the levels of /~-subunit of F1-ATPase in maize dry seed embryos were about 3-fold less than in the 120 h tissue. The levels of both proteins increased during the first 24 h of germination by a factor of 3

Fig. 2. Nucleotide and predicted amino acid sequences of HSP60 cDNAs from A. thaliana and maize and the determined N-terminal protein sequence of mature maize HSP60. Nucleotides are numbered on the margins and ends of the D N A sequence. Vertical lines represent the homology in nucleotide sequence. The presumptive polyadenylation signal sequences are underlined. The predicted amino acid sequences are presented below the nucleotide sequence. Only non-identical residues are indicated in the maize deduced amino acid sequence. The leader sequences are indicated in bold letters. An asterisk indicates the predicted (A. thaliana) or determined (maize) first residue of the mature protein. The determined N-terminal amino acid sequence of mature maize HSP60 (36 residues) is shown between arrows. Several restriction enzyme sites are labeled above (A. thaliana) or below (maize) the nucleotide sequence.

880 FgveaRalml FGNDARVKML FGVEGRASLL FGVEARALML MYRAAASI~%S K A R Q ~ S L A T R Q V G S R L A W S R N Y A A K D I K FGVEARALML ......... M L R L P T V F R Q M R P V S R V L A P H L T R A Y A K D V K F G A D A R A L M L ................................ GADAKEIA FDQKSRAALQ

.GVe.LAdAV RGVNVLADAV KGVETLAEAV KGVEDLADAV RGVEELADAV QGVDLLADAV AGVEKLANAV

kvT.GPkGRn KVTLGPKGRN AATLGPKGRN KVTMGPKGRN KVTMGPKGRN AVTMGPKGRT GVTLGPRGRN

80 VvieqsfGaP VVLDKSFGAP VLIEQPFGPP WIEQSWGAP VVIEQSFGAP VIIEQGWGSP VVLDE.YGNP

Consensus E. coli S. c e r e v i s i a e A, t h a l i a n a Z, m a y s H, s a p i e n s T. a e s t i v u m

81 kvtkDGVtvA TITKDGVSVA KITKDGVTVA KVTKDGVTVA KVTKDGVTVA KVTKDGVTVA KVVNDGVTIA

ksIelkdkf. REIELEDKFE KSIVLKDKFE KSIEFKDKIK KSIEFKDRVK KSIDLKDKYK RAIELANPME

N.GA.Iv.eV NMGAQMVKEV NMGAKLLQEV NVGASLVKQV NVGASLVNRV NIGAKLVQDV NAGAALIREV

A.ktNdaAGD ASKANDAAGD ASKTNEAAGD ANATNDVAGD ANATNDTAGD ANNTNEEAGD ASKTNDSAGD

GTTtatVLar GTTTATVLAQ GTTSATVLGR GTTCATVLTR GTTCDTVLTK GTTTATVLAR GTTTACVLAR

aIftegcksv AIITEGLKAV AIFTESVKNV AIFAEGCKSV AIFTEGCKSV SIAKEGFEKI EIIKLGILSV

aaGmNpmdlr AAGMNPMDLK AAGCNPMDLR AAGMNAMDLR AAGMNAMNLR SKGANpVEIR TSGANPVSLK

160 rGi..aVdav RGIDKAVTAA RGSQVAVEKV RGISMAVDAV RGISMAVDAV RGVMLAVDAV KGIDKTVQGL

Consensus E. coli S. c e r e v i s i a e A. t h a l i a n a Z. m a y s H. s a p i e n s T. a e s t i v u m

161 .eeLkakarp VEELKALSVP IEFLSANKKE VTNLKSKARM VTNLKGMARM IAELKKQSKP IEELERKARP

istseeIaqV CSDSKAIAQV ITTSEEIAQV ISTSEEIAQV ISTSEEIAQV VTTPEEIAQV VKGSGDIKAV

.tISAngd.e GTISANSDET ATISANGDSH GTISANGERE GTISANGERE ATISANGDKE ASISAGNDEL

iG.lia.Ame VGKLIAEAMD VGKLLASAME IGELIAKAME IGELIAKAME IGNIISDAMK IGAMIADAID

KVGkeGViti KVGKEGVITV KVGKEGVITI KVGKEGVITI KVGKEGVITI KVGRKGVITV KVGPDGVLSI

edgktledel EDGTGLQDEL REGRTLEDEL QDGKTLFNEL ADGNTLYNEL KDGKTLNDEL ESSSSFETTV

evvEGMkfDR DVVEGMQFDR EVTEGMRFDR EVVEGMKLDR EVVEGMKLDR EIIEGMKFDR DVEEGMEIDR

240 GyiSPyFitn GYLSPYFINK GFISPYFITD GYTSPYFITN GYISPYFITN GYISPYFINT GYISPQFVTN

Consensus E. coli S. c e r e v i s i a e A, t h a l i a n a Z. m a y s H. s a p i e n s T. a e s t i v u m

241 .ktqk. E . e d PETGAVELES PKSSKVEFEK QKTQKCELDD SKTQKCELED SKGQKCEFQD LEKSIVEFEN

pliL...kKi PFILLADKKI PLLLLSEKKI PLILIHEKKI PLILIHDKKV AYVLLSEKKI ARVLITDQKI

ssiq.ivpvL SNIREMLPVL SSIQDILPAL SSINSIVKVL TNMHAVVKVL SSIQSIVPAL TSIKEIIPLL

Eia.k.rkPL EAVAKAGKPL EISNQSRRPL ELALKRQRPL EMALKKQKPL EIANAHRKPL EQTTQLRCPL

iI.aEDvege LIIAEDVEGE LIIAEDVDGE LIVSEDVESD LIVAEDVESE VIIAEDVDGE FIVAEDITGE

ALatl.iNkl ALATAVVNTI ALAACILNKL ALATLILNKL ALGTLIINKL ALSTLVLNRL ALATLVVNKL

rggikVcAvK RGIVKVAAVK RGQVKVCAVK RAGIKVCAIK RAGIKVCAVK KVGLQVVAVK EGIINVAAIK

320 APgFG.nRKa APGFGDRRKA APGFGDNRKN APGFGENRKA APGFGENRKA APGFGDNRKN APSFGERRKA

Consensus E. coli S. c e r e v i s i a e A. t h a l i a n a Z. m a y s H. s a p i e n s T. a e s t i v u m

321 nlqDiAilTG MLQDIATLTG TIGDIAVLTG NLQDLAALTG NLQDLAIFTG QLKDMAIATG VLQDIAIVTG

geviteelgm GTVISEEIGM GTVFTEELDL GEVITDELGM GEVITEELGM GAVFGEEGLT AEYLAKDLGL

.nlE..t... .ELEKATLED .KPEQCTIEN .NLEKVDLSM .NLENFEPHM LNLEDVQPHD .LVENATVDQ

LGtckkvtv. LGQAKRVVIN LGSCDSITVT LGTCKKVTVS LGTCKKVTVS LGKVGEVIVT LGTARKITIH

kddtvildga KDTTTIIDGV KEDTVILNGS KDDTVILDGA KDDTVILDGA KDDAMLLKGK QTTTTLIADA

gdk.aI.eR, GEEAAIQGRV GPKEAIQERI GDKKGIEERC GDKKSIEERA GDKAQIEKRI ASKDEIQRRV

eqirs.ie.e AQIRQQIE.E EQIKGSIDIT EQIRSAIE.L EQLRSAIE.N QEIIEQLD.V AQLKKELS.E

4OO ttsdYdkEKL ATSDYDREKL TTNSYEKEKL STSDYDKEKL STSDYDKEKL TTSEYEKEKL TDSIYDSEKL

Consensus E. coli S. e e r e v i s i a e A. t h a l i a n a Z, m a y s H. s a p i e n s T. a e s t i v u m

401 qERIAKLsgG QERVAKLAGG QERLAKLSGG QERLAKLSGG QERLAKLSGG NERLAKLSDG AERIAKLSGG

VAV.kvGgas VAVIKVGAAT VAVIRVGGAS VAVLKIGGAS VAVLKIGGAS VAVLKVGGTS VAVIKVGATT

evEvgekkdR EVEMKEKKAR EVEVGEKKDR EAEVGEKKDR EAEVGEKKDR DVEVNEKKDR ETELEDRQLR

vtDAInATrA VEDALHATRA YDDALNATRA VTDALNATKA VTDALNATKA VTDALNATRA IEDAKNATFA

AvEEGivpGG AVEEGVVAGG AVEEGILPGG AVEEGILPGG AVEEGIVPGG AVEEGIVLGG AIEEGIVPGG

GvAll.asre GVALIRVASK GTALVKASRV GVALLYAARE GVALLYASKE GCALLRCIPA GAAYVHLSTY

Idkl.t..an LADLRG..QN LDEVVV..DN LEKLPT..AN LDKLQT..AN LDSLTP..AN VPAIKETIED

480 fDqkiGv.ii EDQNVGIKVA FDQKLGVDII FDQKIGVQII FDQKIGVQII EDQKIGIEII HDERLGADII

Consensus E. coli S. c e r e v i s i a e A, t h a l i a n a Z. m a y s H. s a p i e n s T. a e s t i v u m

481 q.alk. Pa.t LRAMEAPLRQ RKAITRPAKQ QNALKTPVYT QNALKTPVHT KRTLKIPAMT QKALQAPASL

IasNaGvEgs IVLNCGEEPS IIENAGEEGS IASNAGVEGA IASNAGVEGA IAKNAGVEGS IANNAGVEGE

v.vgkl.e ....... IGYdA VVANTVKGGD G...NYGYNA VIIGKLIDEY GDDFAKGYDA VIVGKLLEQD NP..DLGYDA VVVGKLLEQE NT..DLGYDA LIVEKIMQSS S...EVGYDA VVIEKIKESE W...EMGYNA

akgeyv.mv, ATEEYGNMID SKSEYTDMLA AKGEYVDMVK AKGEYVDMVK MAGDFVNMVE MTDKYENLIE

tGiiDP.KV. MGILDPTKVT TGIIDPFKVV AGIIDPLKVI TGIIDPLKVI KGIIDPTKVV SGVIDPAKVT

RtaLvdAasV RSALQYAASV RSGLVDASGV RTALVDAASV RTALVDAASV RTALLDAAGV RCALQNAASV

560 .slltTte.. AGLMITTECM ASLLATTEVA SSLLTTTEAV SSLMTTTESI ASLLTTAEW SGMVLTTQAI

Consensus E. coli S. c e r e v i s i a e A, t h a l i a n a Z, m a y s H. s a p i e n s T. a e s t i v u m

561 vv..Pk.e.a VTDLPKNDAA IVDAPEPPAA VVDLPKDE.S IVEIPKEE.A VTEIPKEEKD VVEKPKPKPK

589 pagaagGgmg gmvgm..mM DLGAA.GG~4G G M G G M C ~ R 4 A . G A . G G M P G GMPGMPG~M ESGAA~ GMVV~4Dy.. PAP~GM...DY.. P~MC,A M G G M G G G M G G G M F . V A E P A E G Q L S V ........

Consensus E. coli S, c e r e v i s i a e A. thaliana z. m a y s H. s a p i e n s T, a e s t i v u m

1 ..................... r .......... n.aaK.ik ................................. MAAKDVK ............ MLRSSVVR SRATLRPLLR RAYSSHKELK MIM~FASNLAS KARIAQN..A .RQVSSRMSW S R N Y A A K E I K

Fig. 3. Comparison of the predicted amino acid sequences of A. thaliana and Z. mays HSP60s with their homologues Cpn60 in E. coil [21], mitochondrial Cpn60s in S. cerevisiae [46] and H. sapiens [23] and the e-subunit of plastid Cpn60 in T. aestivum [21].

The amino acid sequences were deduced from the nucleotide sequences determined for plant HSP60s and their homologues from cDNA and/or genomic clones. Plant mitochondrial leader sequences are shown in bold at amino-terminus. Only the mature coding regions of the various proteins have been compared. Dots represent gaps introduced to maximize the similarity. The G-G-M motifs at the C-terminus are indicated in bold letters. Amino acids are numbered above the amino acid sequence. 'Consensus' codes for homology: capital letters indicate complete homology; lower-case letters indicate > 50% homology; dots indicate < 50 % homology.

881 Table 1. A comparison of the predicted amino acid sequence identities in chaperonins from several organisms. Only the mature coding regions of the various proteins have been compared. The comparisions were made with the introduction of a few gaps to maximize the similarity.

Arabidopsis thaliana Escherichia coli Saccha~vmyces cerevisiae Homo sapiens Triticum aestivum Brassica napus Brassica napus Arabidopsis tbaliana 2 Arabidopsis thaliana 2

cpn60s

A. thaliana I

Z. mays 1

Ref.

mitochondrial bacterial mitochondrial mitochondrial plastid c~ plastid c~ plastid fl plastid e plastid/~

100 56 60 58 44 45 43 44 46

86 55 58 58 44 46 43 43 45

This paper 21 24, 46 23 21 34 34 34 34

1 % Identity: identical residues shared by both proteins when optimally aligned. 2 Only the partial coding region was compared.

to 5. The levels of hsp60 decline during the remaining 120 h whereas the levels of fl-subunit of F1-ATPase remained at the high level (Fig. 5C). Effect of heat shock on HSP60 gene expression

To examine the heat shock response inA. thaliana and maize HSP60 m R N A and protein levels, total

RNAs and mitochondrial proteins were isolated from 5-day old control seedlings and from seedlings that were heat shocked for 3 h at 37 °C. Figure 6 shows that the HSP60 m R N A and protein levels were induced about 2- to 3-fold above basal levels in both A. thaliana and maize upon heat shock.

Discussion

Fig. 4. Southern-blot analysis of t.he A. thaliana gene hybridized with the HSP60 cDNA. Genomic DNA from A. thaliana (10 #g/lane) was digested with Hind III (lane 1), Pst I (lane 2) and Eco RI (lane 3), resolved on 0.7% agarose gels and trans-

ferred to nitrocellulose filters. The filter was hybridized with labeled HSP60 cDNA insert. The hybridization conditions were described in Materials and methods. Hind III-digested lambda DNA was used as molecular weight markers and the band positions are designated on the left of the autoradiograph.

Unlike other organisms, plants have the complexity of having two homologous chaperonins, one located in chloroplasts (plastid Cpn60) and the other in mitochondria (HSP60). Studies on the specificity of anti-HSP60 antiserum indicate that these two homologous proteins are immunologically distinct. In light of this result, we used polyclonal antiserum raised against purified maize HSP60 for screening maize and A. thaliana cDNA expression libraries to isolate positive HSP60 c D N A clones, thus minimizing false positive cDNA clones encoding plastid Cpn60. The identification of the isolated c D N A clones as the full-length HSP60 cDNAs is based on two different criteria: (1) the determined aminoterminal protein sequence of the mature maize protein is in complete agreement with the predicted amino acid sequence of maize c D N A insert and 93~o agreement with the A. thaliana cDNA insert in the corresponding region; (2) the

882

Fig. 5. Northern- and western-blot analyses on the developmental regulation of HSP60 and atp2 (/3-subunit of F 1ATPase) gene expression in A. thaliana and maize during seed germination. Northern: total RNA was isolated from embryos (0 h) or seedlings at different stages of germination (24 to 120 h) and resolved on 1% formaldehyde agarose gels. The RNA was transferred to nitrocellulose filters and hybridized with respective labeled cDNA inserts as described in Materials and methods. A, total RNA from A. thaliana (10 #g/ lane);/3 and C, total RNA from maize (10 #g/lane) hybridized with respective labeled HSP60 cDNA inserts (A, B) or atp2 cDNA insert (C). The sizes of liSP60 and atp2 transcripts are approximately 2.4 kb and 2.1 kb, respectively.Western: mitochondrial proteins were isolated from the same developmental stages of seed germination from A. thaliana (A) and maize (B, C) as described for RNA isolations. The proteins (15 #g/ lane) were resolved on 11% polyacrylamide gels and transferred to nitrocellulose filters. The filters were reacted with anti-HSP60 polyclonaI antibodies (A, B) or anti-/3-subunit of FI-ATPase monoclonal antibodies (C). The sizes of HSP60 and/3-subunit of F1-ATPase are approximately 62 kDa and 55 kDa, respectively.

degree of homology is 43 to 60~o at the amino acid level between maize/A, thaliana HSP60s and their homologues in prokaryotes and in the same or other eukaryotes. In spite of the high conservation of the amino acid sequence between maize and A. thaliana, significant cross-hybridization of cDNAs was only seen with reduced stringency (data not shown). The lack of strong crosshybridization under high-stringency conditions likely reflects numerous third-codon position nucleotide substitutions. Evidently, the analysis of a large group of plant sequences indicates that synonymous codons are used differentially by monocots and dicots, primarily in the use of G or C as the degenerate third base [38].

Fig. 6. Northern- and western-blot analyses on the effect of heat shock on HSP60 gene expression in A. thaliana and maize. Northern: total RNA was isolated from control or heat-shocked 5-day-old seedlings ofA. thaliana (A) and maize (B) and resolved (10 #g/lane) on 1% formaldehyde agarose gels. The RNA was transferred to nitrocellulose filters and hybridized with respective labeled HSP60 cDNA inserts as described in Materials and methods, Western: mitochondrial proteins from A. thaliana (A) and maize (B) were isolated from similar control or heat-shocked seedlings as described for RNA isolations. The proteins (15 #g/lane) were resolved on 11% polyacrylamide gels and transferred to nitrocellulose filters. The filters were reacted with anti-HSP60 antiserum.

HSP60 is an evolutionarily highly conserved protein [35]. Predicted amino acid sequences of maize and A. thaliana HSP60s show high sequence similarities to their homologues in other organisms (Fig. 3 and Table 1). The amino acid substitutions apparently do not greatly alter the function since HSP60 homologues from different organisms can substitute for each other in macromolecular assembly [17, 30, 31]. The overall structure of chaperonin proteins in various species is highly conserved as the alignment of the predicted secondary structures of HSP60 homologues shows similar patterns of e-helix interrupted by/?-sheet regions [23]. Similarity among those proteins begins at residue 35 of maize and 32 of A. thaliana which suggests that the region from methionine (start codon) to the 35th or 32nd residue, respectively, evolved as the leader sequences for mitochondrial targeting [54]. This amino-terminus is rich in basic and hydroxylated residues, a characteristic feature of mitochondrial leader sequences [58]. One notable feature of bacterial and mitochondrial chaperonins is that there is a repeat of a G-G-M motif at the carboxyl-terminus of these polypeptides which is conspicuously absent from the plastid chaperonin (Fig. 3). The significance

883 of this motif is not yet understood. It can be speculated that it is necessary either for the stability of the polypeptide or to serve as a peptide binding site for other polypeptides during the macromolecular assembly process. Various stresses including heat shock induce the synthesis of a set of proteins in all organisms thus far tested [32, 39, 56]. But, for some, their role during normal and/or stress conditions has only recently been realized [27, 43, 53 ]. We report here that heat shock increases HSP60 transcripts and polypeptide levels up to 2- to 3-fold in both maize and A. thaliana. Similar heat inducibility of HSP60 homologues in other organisms has also been reported [16, 35, 46]. Other stress agents such as ethanol, nalidixic acid and H202 are also reported to induce CPN60 expression in cyanobacteria [8]. Although macromolecular assembly seems to be the main function of HSP60 in mitochondria, its participation in stabilizing or protecting disassembled polypeptides under heatshock temperatures is also possible. Developmental regulation of heat-shock proteins during growth at normal temperatures was reported in animals and recently in plants [2, 20, 32, 56]. Extending the previous studies at the protein level in maize [45], we report here that HSP60, both at the mRNA and polypeptide levels, is developmentally regulated in maize and A. thaliana during seed germination. When the levels of HSP60 mRNA and polypeptide were compared between quiescent and germinating embryos, transcript levels increased approximately 5-fold (15-fold compared to 3-fold) more than did the translated products. This discrepancy could be due either to a post-transcriptional modulation of gene expression or to turnover of the polypeptide. To understand the requirement for increased HSP60 during active mitochondrial biogenesis, we investigated the regulation of the fi-subunit of F1-ATPase that participates in mitochondrial oxidative phosphorylation during mitochondrial biogenesis in the early stages of embryo germination. We chose to investigate the F~-ATPase regulation to monitor mitochondrial biogenesis because we know from previous studies that HSP60

is involved in F1-ATPase assembly in maize [44] and yeast [6] mitochondria. As expected, the fisubunit of F1-ATPase, both at mRNA and polypeptide levels, accumulated substantially upon germination compared to levels in quiescent embryos, a result similar to HSP60 regulation at these developmental stages. These data suggest that higher levels of HSP60 are required for the rapid assembly of F1-ATPase during the periods of active mitochondrial biogenesis. A detailed study by Ehrenshaft and Brambl [13] describing the kinetics of the synthesis of FI-ATPase and cytochrome oxidase during maize mitochondrial biogenesis in the early stages of embryo germination also supports our conclusion that germinating embryos undergo rapid mitochondrial biogenesis and therefore, require increased HSP60 levels. While there is good evidence for the function of HSP60 in macromolecular assembly, recent reports in yeast showed that the mitochondrial HSP70 homologue is also involved in this assembly process [25, 52]. The model postulates that the mitochondrial H S P70 homologue accepts the incoming precursor proteins, keeps them unfolded and possibly mediates their transfer to HSP60. However, such a mitochondrial HSP70 homologue is yet to be reported in plants. Given the nature of the distribution of HSPs and their conserved functions, it is most likely that such a model for the involvement of both HSP70 and HSP60 in macromolecular assembly applies to all mitochondria.

Acknowledgements The anti-HSP60 antiserum used in these studies was prepared during the period of an interdisciplinary project supported by the ISU Biotechnology Council in collaboration with Dr R.L. Hallberg. We wish to express our appreciation to Dr R. Brambl for supplying the atp2 cDNA insert, to Dr T.E. Elthon for supplying the anti-fi-subunit F1-ATPase and to Dr Dan Voytas for help with the computer programs and for critically reading the manuscript. We also thank Dr P.J. Rayapati

884

for assisting in the preparation of anti-HSP60 antiserum and Dr L. Tabatabai and the ISU Protein Facility for determining the N-terminal HSP60 protein sequence.

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cDNA clones encoding Arabidopsis thaliana and Zea mays mitochondrial chaperonin HSP60 and gene expression during seed germination and heat shock.

Mitochondria contain a nuclear-encoded heat shock protein, HSP60, which functions as a chaperonin in the post-translational assembly of multimeric pro...
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