Lehle Seeds, Round Rock, Texas, and light-grown seedlings were maintained as suggested by the sup- plier. Pea seeds (Pisum sativum L. cv. Bunting) were.
7610 619
Plant Molecular Biology 31: 619-{i29, 1996. 1996 Kluwer Academic Publishers. Printed in Belgium.
©
AU1, a mitochondrial homologue of the Escherichia coli DnaJ protein Barbara Kroczynska1,3, Rengang Zhou l ,4, Clifford Wood 2 and Jan A. Miemyk l ,* I Mycotoxin Research Unit, USDA, Agricultural Research Service. National Center for Agricultural Utilization Research. Peoria, IL 61604-3902, USA (* authorfor correspondence); 2Department ofBiological and Nutritional Sciences, the University, Newcastle upon Tyne, NEI 7RU, UK; 3Pennanent address: Institute ofBiochemistry and Biophysics, Polish Academy ofSciences, Pawinskiego SA, 02-106 Warszawa, Poland; 4 Pennanent address: Institute ofAgro-Physics, Plant Physiology and Biochemistry, Hebei Academy ofAgricultural Sciences, Shijiazhuang, People's Republic of China
Received 13 February 1995; accepted in revised fonn 27 March 1996
Key words: Arabidopsis thaliana, ATPase activity, Dnal, molecular chaperone, mitochondria, nucleotide sequence, precursor, protein import
Abstract The nucleotide sequence of a cDNA clone from Arabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJI, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains of Escherichia coli Dnal, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis, Arabidopsis appears to have a single at]I structural gene. A single species of mRNA, of 1.5 kb. was detected when Arabidopsis poly(A)+ RNA was hybridized with the atJ] cDNA. The function of at]J was tested by complementation of a dnaJ deletion mutant of E. coli, allowing growth in minimal medium at 44 dc. The AtJ I protein was expressed in E. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ 1 protein was purified to electrophoretic homogeneity. In vitro, recombinant AtJ I stimulated the ATPase activity of both E. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJl contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJl was synthesized in E. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane. Abbreviations: MBP, maltose binding protein; PCR, polymerase chain reaction; Stress70c, the cytosolic member of the 70 kDA family of stress-related proteins
Introduction The heat shock regulon of Escherichia coli consists of a group of proteins whose synthesis is transiently increased when cells are exposed to elevated temperature or other environmental insults [21]. The heat shock response is one of the most highly conserved The nucleotide sequence data reported will appear in the EMBL. GenBank and DDB] Nucleotide Sequence Databases under the accession number U 16246.
biological responses. Presumably, many heat shock proteins function by increasing the ability of cells to survive conditions that disrupt native protein structure. Among E. coli heat shock proteins are the products of the dnaK, dnaJ and grpE genes. The DnaK protein is a member of the Stress70 superfamily of proteins [12]. The Stress70 proteins function as molecular chaperones, and have a low level endogenous ATPase activity [2]. Dnal and GrpE proteins can function as co-
Supplied by U.S. Dept of Agriculture National Center for Agricultural Utilization Research, Peoria, Illinois
620 chaperones, and together stimulate the ATPase activity of E. coli Stress70 many-fold [19]. The interactions among DnaK, DnaJ and GrpE have been examined in detail by Zylicz and associates. DnaJ stimulates the hydrolysis of ATP by E. coli DnaK, while GrpE proteins function as nucleotide exchange factors, promoting release of bound ADP [19, 20]. Other reports, however, suggest that functional relationships among DnaK, DnaJ and GrpE might be more complex. In some instances, DnaJ can increase the avidity of interactions between DnaK and target proteins [14], and there have been reports that GrpE can either stabilize [33] or destabilize [14] interactions between DnaK and target proteins. It was recently reported that DnaJ can itself bind to nascent polypeptides and maintain them in an unfolded or loosely folded conformation [14]. This raises the possibility that, in addition to functioning as a cochaperone, DnaJ might function independently as a molecular chaperone. Using a plant-derived in vitro membrane translocation system, it was demonstrated that cytoplasmic Stress70 can act as a molecular chaperone for a secretory precursor [25]. Thus, interactions between molecular chaperones and co-chaperone proteins appear to have been highly conserved during evolution [5, II, 13]. The experiments presented herein were undertaken as an initial step in the analysis of the roles of higher-plant homologues of DnaJ in protein folding and organelle biogenesis.
Radioisotopes [a- 32 P]-dCTP (29.6 TBq/mmol) was from DuPont-
NEN, Wilmington, DE, USA. Pro-mix, a mixture of [ 35 S]-Met and [35 S]_Cys (37 TBq/mmol), was from Amersham International, Little Chalfont, Bucks, England. Biological materials
The maize (Zea mays L., inbred A636) endosperm cell cultures have been previously described [24]. The Al cell line was used for preparation of Stress70c. Seeds of WT-2 A. thaliana, ecotype Columbia, were from Lehle Seeds, Round Rock, Texas, and light-grown seedlings were maintained as suggested by the supplier. Pea seeds (Pisum sativum L. cv. Bunting) were a generous gift of Batchelors' Foods, Worksop, Notts, England, and germinated as previously described [23]. SURE E. coli host cells, Stratagene, La Jolla, CA, were used for propagation of sequencing templates, while SGl611 or PR745 cells were used as the host for heterologous protein expression. The dnaJ- strain PKlO2 [17] was generously supplied by E.A. Craig, University of Wisconsin. Competent cells were prepared, and then transformed, using standard methods [22]. Transformed bacteria were grown at 37°C on LB plates containing 200 fLg/ml ampicillin. Both stock and transformed PKlO2 cells were maintained at 30°C. Plasmids
Materials and methods Reagents
All buffers were from Research Organics, Cleveland, OH. Unless otherwise noted, DNA modifying enzymes were from New England BioLabs, Beverly, MA, and were used according to the manufacturers' recommendations. The purified E. coli DnaK, DnaJ and GrpE proteins were purchased from Epicentre Technologies, Madison, WI. Malachite Green carbinol base was from Aldrich Chemical Company, Milwaukee, WI. Other biochemicals were from the Sigma Chemical Company, St. Louis, MO, and were of the highest purity available.
The plasmid pJAMI50, previously designated 23f2t7 by the Michigan State University Arabidopsis Genome Project, is a Sail x NotI cDNA fragment directionally cloned into the vector pZLl. The general-purpose vector pUCI9, Boehringer Mannheim, Indianapolis, IN, was used to subclone fragments for sequencing. The E. coli vector pMAL-c2, for expression of heterologous proteins as chimera with the MBp4, was from New England BioLabs, Beverly, MA. In order to construct a plasmid encoding the MBPAtJl chimeric protein, the insert from pJAM150 was transferred into pMAL-c2. First, pJAM 150 was digested with BsrGI and HindIII and the insert separated from the vector. The oligonucleotides 5'CGATCGAGGGAAGGATGCGAAGATTCAACTGG GTTCTGCGGCAT-3' and 5'-GTACATGCCGCAGA ACCCAGTTGAATCTTCGCATCCTTCCCTCGATC GAGCT-3' were synthesized, purified, and annealed. The plasmid pMAL-c2 was digested with Sad and
621 HindIII, and purified. The original insert, annealed oligonucleotides, and digested vector were then mixed and ligated [22]. This construction was designated pJAM165. To express AtJl in E. coli directly, rather than as a chimera, the malE gene was deleted from pJAM165. The oligonucleotides 5'-AATCTATGGTCCTIGTIG GTGAA-3' and 5'-TACGCTICTAAGTIGACCCAA GA-3' were synthesized. These oligonucleotides plus pJAM 165 previously linearized by digestion with Sad, were used in the PCR. The product of PCR was purified, treated with the Klenow fragment of DNA polymerase I, then ligated [22]. The resultant plasmid was designated pJAM 166. A plasmid was designed to allow high level direct expression of AtJ 1 in E. coli and at the same time simplify purification. The pJAM166 construction was digested with HindIII and HpaI, and the vector separated from the insert. The oligonucleotides: 5'-T ACATCACCATCACCATCACTAAAAGCTIGGG-3' and 5'-CCCAAGCTITIAGTGATGGTGATGGTGA TGTA-3' were synthesized, purified, annealed, and then digested with HindIII. HpaI cuts within the stop codon of the original AtJ 1 reading frame. The oligonucleotides include a substitution of A for T in the original stop codon, converting it into a codon for Leu, followed by six His codons. The HindIII x HpaIdigested pJAM 166 was then mixed with the synthetic dsDNA and ligated. This construction was designated pJAM 167. All constructions were verified by sequencing. Sequence analysis
Sequencing was performed using an Applied Biosysterns Taq DyeDeoxy Terminator Cycle Sequencing kit and a Model 370A Applied Biosystems Automated DNA Sequencer. The sequences of internal primers were based upon 3'-proximal regions of previously determined sequences. 20-Mer sequencing primers were prepared using the Applied Biosystems Synthesizer. DNA sequence analysis and manipulation, and comparison with published sequences, were accomplished using the program PC/GENE, from intelliGenetics, Mountain View, CA. Nucleic acid analyses
Total RNA was isolated from various tissues of A. thaliana using the materials and protocol supplied by Clontech, Palo Alto, CA, with the Extract-A-Plant
RNA isolation kit. Poly(A)+ RNA was separated from total RNA using the mRNA Separator kit from Clontech laboratories. Electrophoresis of RNA through formaldehyde-containing gels, transfer onto Amersham Hybond N nylon membranes, and hybridization with DNA probes were according to established methods [22]. Genomic DNA was isolated from Arabidopsis leaves, restriction digested, separated by agarose gel electrophoresis, and transferred to nylon membranes by standard procedures [22]. The insert from pJAM 150 was labeled with [3 2p]-dCTP using the Random Primer DNA Labeling Kit from BioRad Laboratories, Richmond, CA. This same probe was used to detect RNA on northern blots and DNA on Southern blots. Complementation ofa dnaJ- E. coli mutant
Single colonies of PKI 02 cells from stock plates were used to innoculate small flasks containing 20 ml of LB medium containing 200 /lg/ml ampicillin. The flasks were incubated overnight at 30°C with vigorous shaking. The overnight cultures were diluted into flasks containing 50 ml of M9 medium [22] plus 200 /lg/ml ampicillin and 0.3 mM IPTG, such that the A600 was 0.01. The flasks were then incubated with vigorous shaking at either 37°C or 44 0c. Aliquots were periodically removed to determine the A600. Protein purification
Starting with a cytosol fraction [30], maize endosperm Stress70c was purified to electrophoretic homogeneity by a combination of affinity and anion exchange chromatography [36]. Protein levels were quantitated using the dye-binding assay [4], with BSA as the standard. Initially, recombinant AtJ 1 protein was synthesized in E. coli as a chimera with the MBP. An overnight culture of transformed SG1611 cells was diluted into fresh LB medium, containing 200 /lglml ampicillin, to an A600 of 0.05. Flasks were then incubated at 37°C with vigorous shaking until the A600 was ca. 0.5. At this stage, IPTG was added to a final concentration of 0.3 mM in order to induce synthesis of the fusion protein. Cells were grown for an additional three hours, then harvested by centrifugation, and frozen. The frozen cell pellet was thawed by resuspension in amylose column buffer; 10 mM Tris-HCI pH 7.4, containing 200 mM NaCI and 1 mM EDTA. The resuspended cells were disintegrated by sonication for a total of2 min at a setting of five, using the microtip of a Heat Systems-Ultrasonics, (Farmingdale. NY), mod-
622 el W-380 Ultrasonic Processor. The sonicate was clarified by centrifugation, then applied to an amylose resin column (New England BioLabs). After washing with column buffer, bound proteins were eluted with column buffer containing 10 roM maltose. Eluates were concentrated to ca. I mg/ml using Amicon microconcentrators, then treated with Factor Xa for 12 h at room temperature. The cleaved fusion protein was subjected to a second round of amylose column chromatography. The AtJ I protein, which was present in the column flow-through volume of the second chromatographic run, was concentrated and dialyzed overnight against FPLC column buffer; 200 volumes of 20 roM Tes-NaOH pH 7.0, containing 0.1 mM EDTA. The dialysate was passed through a 20 pm filter, then loaded onto a Mono Q HRS/5 anion-exchange FPLC column. The column was washed with equilibration buffer, and bound proteins eluted with a linear gradient of 0 to 500 mM KCI in column buffer. Column fractions were evaluated by SDS-PAGE, and AtJI -containing fractions combined and concentrated. The AtJ I protein eluted from the column at ca. 100 roM KCI. SS]-AtJI(H6) was prepared by incubating E. coli PR745 , transformed with pJAMI67, with [3SS]-Promix. An overnight culture was diluted with fresh LB plus ampicillin medium to an A600 of 0.05. The cells were then grown at 37°C with vigorous shaking to an ~oo of 0.5. At that time, IPTG was added to a final concentration of 0.3 mM along with 7.4 MBq of eSS]-Pro-mix and the cultures were incubated for an additional two hours. Cells were collected by centrifugation, then washed once by resuspending in 100 roM sodium phosphate pH 8.0, and re-pelleting. Washed cells were suspended in 5 mL of sodium phosphate and disintegrated by sonication. Cellular debris were removed by centrifugation and the supernatant was used for purification of [3s S]-AtJI(H6)' The clarified supernatant was loaded onto a 10 ml column of Ni2+ NTA-agarose (Qiagen, Chatsworth, CA) previously equilibrated with 100 mM Sodium phosphate pH 8.0. The column was successively washed with 100 mM sodium phosphate pH 8.0, 500 KCI in sodium phosphate pH 8.0, and 100 sodium phosphate pH 6.0. The e SS]-AtJl(H6) protein was then eluted with 20 roM imidazole, pH 6.0. Purity of samples was evaluated by SDS-PAGE plus Radioanalytic Imaging [25]. If additional purification was necessary to achieve electrophoretic homogeneity, a second round ofNi2+ -NTA chromatography was conducted with the final elution using 100 roM sodium phosphate pH 5.5.
e
ATPase assays ATPase activity was quantitated using the malachite green procedure [1, 10]. Absorbance of the phosphomolybdate plus malachite green complex was measured at 600 nm using a Cambridge Technologies Model 700 microplate reader. Standard curves were constructed using KH2P04 in 5% HCI04, and samples were deproteinized by adding HCI04 to a final concentration of 5% (v/v). A typical ATPase assay mixture contained buffer, salts, 2 mM DTT, 0.1 roM ATP, and 2 pg of purified Stress70 protein in a final volume of 0.1 ml. The assays of DnaK contained 30 mM TES-NaOH buffer, pH 7.5, plus 20 roM KCI, 50 roM NaC!, and 7 roM MgCh, while those of maize Stress70c contained 100 roM BisTris, pH 6.0, plus 20 roM KCI and 1 roM MgCh. After 10 min at 40°C, reactions were stopped by adding 0.01 ml of 55% (v/v) HCI04. Reaction mixtures were incubated on ice for 10 min, then centrifuged for 3 min at maximum speed using a Eppendorf 5415 microcentrifuge. Samples of0.08 ml from each supernatant were taken for measurement of ~.
Isolation ofmitochondria Purified intact mitochondria were isolated from the cotyledons of germinating pea seedlings by rate-zonal sedimentation exactly as previously described [34]. The final preparations were resuspended in buffered osmoticum without BSA.
Impon ofAtlI by pea mitochondria Import reactions contained ca. 0.2 mg of mitochondrial protein and 5 x 106 dpm [3sS]-AtJl(~) in a final volume of 0.2 ml. Typically the following components were present at the indicated final concentrations: sorbitol (250 roM), KCI (50 mM), Hepes buffer pH 7.2 (10 roM), ATP (2 mM), GTP (2 mM), . ADP (0.01 mM), NADH (0.5 roM), L-malate (l mM), NaH2P04 (5 mM), and MgCh (5 roM). Other Krebs cycle intermediates were tested as respiratory substrates, but at equal concentrations none worked as well as malate. In some instances the ionophore valinomycin was added to a final concentration of 2 pM. Reactions were incubated for one hour at 25°C. After reactions were completed, samples were incubated on ice. In some instances proteinase K was added to a final concentration of 20 pg/ml, with or without 0.1 % Triton X-100. After 20 min, PMSF was added to a
623 final concentration of I mM. The tubes were then centrifuged and the pellets processed for SDS-PAGE and Radioanalytic Imaging [25].
CAAAAAC=C=C==AGC'1"I'1'GAA'I'rCACAAlaCTC
-1
ATGCGAAGATTCAA===AC1.AGC'1'CGAAGAC':l'l.'1"mA=
60 20
A'l'CGGATTG~TACA'I'rCAC:CCTACAGG'I'
120 40
ATMACAA==AATTACTATGA~CTACA INN S 5 h R H Y X L G 5 P KAT
180 60
M R R Y I
G
L
N W V L R B V Q A R R T Y
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Results and discussion Sequence comparison ofAtlI with DnaJ and eukaryotic homologues
0
S A
BAT
G
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CGGGAGGAGATTMAMATCAT'I'rCA1'GAGCTTGc:cMAMATTCCACCCTGATACAAAT BEE I K K 5 f H E L A K K F H P P T H
HO
AGMATAA~TAAGAGAGGCATATGAGA= B H H P S h K B K f Q t I B E h X E T L
300 100
GGMA~TATGATAAGCTGCAG'rATCGGAATTCGlaTTA=A
360 120
AATAATGA==TT~TACCAG'rCCAA'l"l'TC'rCGGAT
420 140
A=CCACAAla=cTGAGATA1"1"MAGAACAATCAGATAAAACCTGATATTCCG
480 160
G
N
s
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G G 0
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TFHKIYPEIFENNQIKPDIR
The results from an initial partial sequence analysis suggested that the DNA insert in pJAMI50 might encode a homologue of the E. coli Dnal protein. The complete sequence of the insert was subsequently determined, both by subcloning internal fragments and using sequencing primers based upon prior results. Translation of the insert revealed an open reading frame of 368 amino acid residues that would encode a protein of 41 471 Da molecular weight with a pI of 9.2 (Fig. I). Dnal proteins have three characteristic primary sequence domains. The N-terminal region of ca. 70 residues is termed the J domain [3]. Distal to the J domain is a region rich in glycine and phenylalanine residues, the G or GF domain. Downstream of the GF domain is a cysteine-rich C domain, typically containing four repeats of the CxxCxGxG motif, where x can be any amino acid residue. While all Dnal homologues contain the J domain, the occurence of the GF and C domains is variable [3, 26]. The deduced amino acid sequence of AtJ I contains within the N-terminal region a sequence with high homology to the J domain of Dnal homologues (Fig. I and 2A). Sequences of all of the Dnal homogues identified to date contain a conserved motif, YIFHPD. It was proposed that this sequence forms a turn between two putative alphahelical domains ofDnal, and that this region is essential for physical interaction with DnaK [35]. This proposed turn forming domain is present within the J domain of the AtJI sequence, F7s HPD (Fig. I). While the GF-domain is absent, the AtJ I sequence contains one perfect copy and three imperfect copies of the signature sequence of the Cys-rich domain, CxxCxGxG (Fig. I). It has been noted that these repeated sequences bear some resemblance to the zinc finger motif of DNA-binding proteins [5], and that Dnal can bind to DNA [41]. It is not yet clear, however, that the zinc finger-like sequence is related to the biological function of Dnal, and it is not present in all Dnal homologues [ef. 26]. The average overall sequence identity among Dnal homologues is 32% (Fig. 2B). When sequence simi-
GTGGAA==CCAAGCTGCAGAAGGGTGCACAAAA==
VEL
S
L S
L SEA
A E G C T
K R L S
F
80
540
180
~TGf'T~~Cli?~rGF'FEN9:""1~~A~TGFXCAf
600 200
A;c"r7Fer:rgCh~~~CT~'l"l]CT~Cl~llLA~~T
660 220
~'7SC"$GA~M~~CArrArr~T~~~AgA
120
~AiA~OOf'~~~'F~~
780 260
GACTCTGAAGCTACAATCACAA=A=AATGTAAG'M'CAAGAACAAGTCAA ESE A T I T I V GAG N v S S R T S Q
840 280
CCTGGGAACTTG'l'ATATCAAACTMAGGTTGCTAATGATTCAACT'I'rCACTAGGGATGGC
900 300
TCAGATATATATGTGGATGCTAATATTAG=ACACAAGCTATTTTGGGGGCCAAAGTT
960 320
GTGGTGCCAACACTTTCAOOCAAGATACAGCTAGATATACCAAAGGGGACTCAGCCTGAT
1020
CAACTTC'l""1'GllftAAGAGCCAAAGGAC'rAccaAAGCAAGGCl11 II I l'G'%AGATCATCG
1080 360
AGATCAGTA=C:CCTTCCG=AA=CCTACTGAAGTGAATGAA~
1140 368
TATACTGGAAGAGTTTCCAAAGGAAGAAATCAACAATGAGTTGA==CTGAAGG AAGTTGGTGGAATCTAACGGGTCCTCAGATCA=CGA=AA== GGCCCTATTGTTGAGCAGGTTAATGGGATGAAGCCACCACGAGAGATGAAAGATAAAAGC ATTCAACAGAAGACAAATGAAGACTAATTTCATCTATTGCTGGTAGAACCCAAAGCTCAC CAGCATAACAGAGA=CATTGTTTGACCTCATAA=ATTACAAACTCA=ATGT GAATAATAAGAGACA'I'CCAACAAGttAAGC'l"GAAATAAAGJ, a..l J,GA ill"" ill ... UGTCCT
1200 1260 1320 1380 1440 1500 1521
P G N L Y I
SOL
Y v
V V P T
Q L L v
0
L S
K L K V AND
A N I
G K I
S
F T
Q L 0
L R A K G L P
R S V C S
L
Q
I
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R 0
F
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0
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'I"TAJ.ll& J,),. U),A aa ;\GGGC
240
340
Figure I. The nucleotide and deduced amino acid sequences for the AtJ! protein. The J-domain is underlined. and the FHPD ~-turn motif is double underlined. The CxxCxGxG repeat sequences of the C-rich domain are boxed. The nucleotide sequence has been deposited in GenBank under the accession number U! 6246.
larity is considered, this values increases to 46%. The sequence homology estimates are remarkably similar, considering that comparisons span the distance from E. coli to man and includes both cytoplasmic and mitochondrial proteins. The J domain is the source of much. of the sequence homology in comparisons among the sequences of Dnal homologues (Fig. 2A). It is noteworthy that the deduced amino acid sequence of AtJ I is no more similar to mitochondrial than cytoplasmic sequences (Fig. 2B). Expression ofAtlI in A. thaliana When poly(A)+ RNA from Arabidopsis leaves was separated and hybridized with the AtJI cDNA probe, a single message was detected (Fig. 3). The band correspond to a size of 1.5 kb. This is slightly larger than the size reported for other plant Dnal homologues [e.g. 28, 37, 39]. Essentially identical signals
624 A. AtJl AtJ2
•16EI ••• PItT •• P .DL •• -AY!QtALJ:.N ... l"GGDPElIEQELAQ------s .. V •• DP .It •• I •.
DnaJ
••• EI •••• ItT .EER •• R.AXltR•• M. Y•.• R.QGDltE.EA •. It. n •••• V. TIl. QIt.AA •• QY
MdJ1
Pc> .DT •• LltltS •• GA •.•• AYYlt •••• Y ••• I .ltEPDAE. - .• BOLON •.• I • 5OETlt. QQ •• QF
Hsp40
.5
Yd..n
F, .DI •• GV.T •• DV •••• AYRItC.L. Y ..• 1t.PSEEA.EltF- •• ASAE-- .. I •• DP .K.DI .•
.QT •• LARG.5O •• T.RAYRRQ.LRY ••. K.ltEPGAEEltF- .• :IA-- .... V .• DP.It •• :IF.
B. DnaJ
MdJ1
46(58)
35(49)
31(45)
22(36)
34 (49)
32(46)
31(49)
MdJ1
24(36)
33 (46)
38(55)
DnaJ
31(46)
35(48)
AtJ2
23(38)
AtJ"l
AtJ2
Yd..n
25(38)
Hsp40
Hsp40 37(54)
Figure 2. Analysis of the deduced AlII amino acid sequence. A. Alignment of the J-domain sequence of AlII with those of E. coli DnaJ ([26]; GenBank accession number MI2565). Homo sapiens Hsp40 ([26]; DI7749). A. tfuIliana AlI2 ([37]; L36133) and S. cerevisiae Ydjl ([16];
X56560) and MdJl ([31]; 228336). Periods have been placed to indicate identity with AlII. Spaces. indicated with a dash. have been insened to maximize homology. B. Overall sequence comparison ofDnaJ homologues. Values are positional amino acid identity. with similarity values in parenthesis.
kb.
Southern analysis ofatJ!
9.49 7.46 4.40 2.37 1.35 -
----
