From the Department of Horticulture, Viticulture and
Oenology, The University of Adelaide, Waite Campus, PMB1, Glen
Osmond, South Australia, 5064, Australia, the ** Australian Wine
Research Institute, Glen Osmond, South Australia, 5064, Australia, and
the § School of Biochemistry, La Trobe University, Bundoora,
Victoria, 3083, Australia
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ABSTRACT |
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We previously reported the cDNA cloning and characterization of a mammalian mitochondrial GrpE protein (~21 kDa, mt-GrpE#1) and now provide evidence for the presence of distinct cytosolic (~40 kDa), microsomal (~50 kDa), and additional mitochondrial (~22 kDa, mt-GrpE#2) GrpE-like members. While a cytosolic GrpE-like protein has recently been identified, the demonstration of both a microsomal and a second mitochondrial GrpE-like member represents the first in any biological system. Investigation of the microsomal and two mitochondrial GrpE-like proteins revealed that they bound specifically to Escherichia coli DnaK, and the complexes formed were not disrupted in the presence of 0.5 M salt but were readily dissociated in the presence of 5 mM ATP. The functional integrity of mt-GrpE#1 and #2 was verified by their ability to specifically interact with and stimulate the ATPase activity of mammalian mitochondrial Hsp70 (mt-Hsp70). Analysis of the cDNA sequences encoding the two mammalian mitochondrial GrpE-like proteins revealed ~47% positional identity at the amino acid level, the presence of a highly conserved mitochondrial leader sequence, and putative destabilization elements within the 3'-untranslated region of the mt-GrpE#2 transcript which are not present in the mt-GrpE#1 transcript. A constitutive expression of both mitochondrial GrpE-like transcripts in 22 distinct mouse tissues was observed but possible different post-transcriptional regulation of the mt-GrpE#1 and #2 transcripts may confer a different expression pattern of the encoded proteins.
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INTRODUCTION |
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Molecular chaperones of the 70-kDa heat shock protein
(Hsp70)1 family function in a
diverse number of vital cellular processes including protein folding,
translocation of proteins across membranes, proteolysis, regulation of
the stress response, and activation of signal transduction molecules
(reviewed in Refs. 1-6). Members of the Hsp70 family have been found
in all organisms examined, and representatives from distantly related
organisms often display a high degree of sequence similarity. For
example, at the amino acid level the prokaryotic Escherichia
coli Hsp70 member (DnaK) exhibits ~51% positional identity with
its higher eukaryotic mitochondrial homologue (mt-Hsp70) (7). Central
to the function of Hsp70 members as molecular chaperones is a weak
ATPase activity which, at least in the case of E. coli DnaK,
is synergistically stimulated up to 50-fold by the heat shock proteins
DnaJ and GrpE (8). DnaJ and GrpE appear to regulate the ability of DnaK
to bind and stabilize unfolded proteins before their release in a
reaction requiring ATP hydrolysis (9). It has been shown that DnaJ
stimulates the hydrolysis of DnaK-bound ATP which allows DnaK (in the
ADP-bound state) to interact more strongly with unfolded proteins. GrpE then acts as a "nucleotide exchange factor" to recycle DnaK into an
ATP-bound state, thereby permitting the efficient release of substrate.
Recently, the crystal structure of E. coli GrpE bound to the
ATPase domain of DnaK was determined (10). This confirmed earlier
studies that a dimer of GrpE binds asymmetrically to a single DnaK
molecule in the absence of ATP (11-13). GrpE is proposed to facilitate
release of DnaK-bound ADP by essentially wedging apart the DnaK ATPase
domain, thus weakening the grasp of DnaK on ADP. Furthermore, a long
-helical region of GrpE is believed to extend to the
C-terminal substrate binding domain of DnaK where it could possibly
mediate an interdomain communication necessary for substrate release.
Thus, DnaK/DnaJ/GrpE appear to function in a mechanistic fashion as a
molecular "chaperone team" which, in one form or an other, may
operate in all compartments of eukaryotic cells.
Homologues of DnaK and DnaJ have been identified in several major compartments of the eukaryotic cell, including the cytosol, nucleus, endoplasmic reticulum (ER), mitochondria, and chloroplasts, and in many cases these chaperones exist in multiple isoforms (reviewed in Refs. 1-6). In comparison, eukaryotic homologues of GrpE have been detected only in mitochondria (14-18) and possibly chloroplasts (19), but in no case have isoforms been detected. While a chloroplast GrpE-like protein has been inferred through cross-reactivity with antibodies to E. coli GrpE (19), mitochondrial GrpE homologues from Saccharomyces cerevisiae (Mge1p, Yge1p, GrpEp, and mGrpE), Drosophila melanogaster (Droe1p), and mammals (mt-GrpE#1) were identified by their abilities to bind specifically to Hsp70 members, and the complexes formed are not disrupted in the presence of 1 M salt but readily dissociate in the presence of 5 mM ATP (15, 17, 18). Such a specific interaction was first observed between the E. coli members of GrpE and DnaK (20).
It is somewhat surprising then that the role of a GrpE-like protein may not be required for the proper functioning of Hsp70 homologues outside mitochondria and chloroplasts while DnaJ homologues exist in all additional compartments that contain Hsp70 members. Indeed, it is perhaps more likely that the three-component chaperone team of Hsp70, DnaJ, and GrpE is conserved in numerous compartments of eukaryotic cells, but that sequence similarities have been too low to allow facile identification through comprehensive searches of data bases (21). Thus, as opposed to members of the Hsp70 (DnaK), Hsp60 (GroEL), and Hsp10 (GroES) families from rat mitochondria and E. coli which exhibit 51, 49, and 45% positional identity at the amino acid level, respectively, the GrpE family exhibits only 21% positional identity, and comparison of 20 GrpE sequences identified only six invariant residues (21). Another likely reason for the paucity of identified GrpE-like proteins is their very low abundance in most systems (i.e. Mge1p and mt-GrpE#1 comprise ~0.03% of the total soluble mitochondrial proteins) (21, 22). Taken together, the low degree of sequence similarities and the low abundance of at least some GrpE members calls for both a sequence based and a functional approach to search for new GrpE-like chaperones. Indeed, very recently a cytosolic/nuclear located Hsp70/Hsc70-associating protein (Hap) (alternatively named BAG-1 or RAP46) with GrpE-like activity but a seemingly unrelated primary structure has been reported (23-25). Alternative translation initiation from a single Hap transcript in both human and mouse cells is believed to generate either a cytosolic ~36- and ~32-kDa Hap protein, respectively, or a ~50-kDa Hap protein with an N-terminal extension thought to be important in nuclear targeting (26). The finding of a cytosolic/nuclear-located protein with GrpE-like activity lends further support to the supposition that Hsp70 members operate as components of DnaK/DnaJ/GrpE-like chaperone teams irrespective of the organism or cellular location. Given the presence in the endoplasmic reticulum of both DnaJ-like members (Sec63 and Scj1p) and Hsp70-like homologues (BiP and Lhs1p), it is therefore not unlikely that microsomal proteins with GrpE-like activity exist (reviewed in Refs. 1-6).
In this study we have now further explored the complexity of the mammalian GrpE complement. We provide evidence that mammalian cells, in addition to their previously identified mt-GrpE#1 and cytosolic/nuclear located Hap members, contain a second distinct mitochondrial GrpE member (mt-GrpE#2) which functions as a co-chaperone for mt-Hsp70, plus a microsomal ~50-kDa protein which may serve as a partner for ER BiP or an as yet unidentified Hsp70 member.
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MATERIALS AND METHODS |
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DnaK-affinity Chromatography and Immunological
Techniques--
Preparation of a DnaK-affinity column and subsequent
chromatography was described previously (17). Isolation of bovine liver organelles was performed on sucrose density gradients by removing a
broad region of the gradient where mitochondria predominantly reside
with significant amounts of endoplasmic reticulum and peroxisomes being
present.2 Soluble protein
extracts were then prepared from the purified organelles. Following
DnaK affinity chromatography, 100 µg of the 5 mM ATP
eluate (predominantly comprised of bovine mt-GrpE#1) was mixed with an
equal volume of Freund's complete adjuvant (Life Technologies, Inc.)
and injected subcutaneously into a 7-month-old New Zealand White
rabbit. The rabbit was boosted, three times at 6-week intervals, with
approximately 50 µg of bovine mt-GrpE#1 excised from SDS-PAGE gels,
developed into a slurry, and mixed with an equal volume of Freund's
incomplete adjuvant as described by Harlow and Lane (28). Ear bleeds
were performed 10 days after the third boost, and serum containing
polyclonal antibodies to mt-GrpE#1 was collected and processed (28).
The serum was supplemented with 0.02% (v/v) sodium azide and stored at
70 °C. Dilutions of 1 in 10,000 were used in immunostaining
analysis.
Stress Treatments, Metabolic Labeling, and Fractionation of Tissue Culture Cells-- The growth of rat hepatoma (H4) cells in the presence of the amino acid analogue L-azetidine-2-carboxylic acid, the conditions for heat shock, and the isolation of 35S-labeled cytosolic and mitochondrial proteins were as described previously (21). A crude cellular lysate was prepared by hypotonic lysis and Dounce homogenization. Cellular membranes and nuclei were removed as a pellet following a 754 × g centrifugation, and a crude mitochondrial pellet was obtained by recentrifugation of the supernatant at 10,000 × g. The remaining supernatant represented the cytosolic and microsomal fraction. Mitochondria were purified further on sucrose gradients and following incubation with 0.5% (w/v) Triton X-100 reduced (Sigma) at 4 °C for 1 h, a soluble mitochondrial lysate was obtained after centrifugation at 80,000 × g for 1 h.
Nucleotide Sequence Analysis-- The verified nucleotide sequence encoding mouse mt-GrpE#2 was obtained by sequence analysis of two independently isolated expressed sequence tags (ESTs) from the I.M.A.G.E. Consortium Lawrence Livermore National Laboratory cDNA clones collection (I.M.A.G.E. Consortium CloneIDs 482996 and 478162 corresponding to GenBankTM accession nos. AA060861 and AA049605, respectively; see "Results" and "Discussion") (31). Both strands of the two clones were sequenced using the dideoxynucleotide chain termination procedure of Sanger et al. (32).
Southern Blot Analysis-- Rat genomic DNA (10 µg) was digested with the appropriate restriction enzyme (40 units at 37 °C for 8 h) and electrophoresed in a Tris acetate-EDTA (TAE)-buffered 0.8% (w/v) agarose gel. The digested DNA was transferred onto a HybondTM-N+ nylon membrane (Amersham Pharmacia Biotech), and the blot was prehybridized for 6 h at 65 °C in 5× SSC, 0.5% (w/v) SDS, 100 µg/ml sheared salmon sperm DNA, and 5× Denhardt's solution (0.1%(w/v) FicollTM 400, 0.1% (w/v) polyvinylpyrrolidone, and 0.1% (w/v) bovine serum albumin). The membrane was probed with a 32P-labeled mouse mt-GrpE#2 cDNA fragment (nucleotides 65-690, Fig. 1), then stripped by incubation in 0.5% (w/v) SDS at ~90 °C for 10 min and reprobed with a 32P-labeled rat mt-GrpE#1 cDNA fragment (GenBankTM accession no. U62940, nucleotides 96-733). The denatured probes were added to the prehybridization solution and hybridizations were carried out for 12 h at 65 °C. Final washes were performed for 10 min at 65 °C in 0.1× SSC supplemented with 0.1% (w/v) SDS, and the blots were analyzed using a Storm PhosphorImager and Image QuaNT software (Molecular Dynamics).
Expression of Mouse mt-GrpE#2, Rat mt-GrpE#1, and Rat mt-Hsp70 in E. coli-- The cDNA coding regions specifying the mature translation products of mouse mt-GrpE#2 (579 bp), rat mt-GrpE#1 (570 bp), and rat mt-Hsp70 (1899 bp, GenBankTM accession no. S75280) were amplified with VentR®DNA polymerase (New England BioLabs) utilizing specific primer pairs in a 30-cycle polymerase chain reaction protocol (94 °C for 60 s, 55 °C for 90 s, and 72 °C for 90 s). The primer pairs used were: for mouse mt-GrpE#2 (5'-GTTAACATATGAGCACTGCCACCCAAAGAACT-3' + 5'-AGCCGGATCCTTAGAGTCTTCTCTGAGACTCTAC-3'), for rat mt-GrpE#1 (5'-CCATTGGGATCCTAAGGAGGTTAACATATGTGCACAGCTACAAAACAAAAG-3' + 5'-CGGAATTCAAGCTTGGATCCTTAAGCGTCCTTCACCACCCCCAC-3'), and for rat mt-Hsp70 (5'-ATGGATCCATATGGCGTCAGAAGCAATCAAGGGTGC-3' + 5'-TTGGATCCTTAATGATGATGATGATGATGAGAACCCCGCGGAACTAA-3'). PCR products were digested with NdeI and BamHI, then ligated individually into the same predigested sites of the pET-14b vector (for mt-GrpE#1 + #2) or pET-3a vector (for mt-Hsp70) as specified in the manufacturer's manual (4th Ed., Novagen). Note that the mt-Hsp70 construct was designed to remove a preexisting N-terminal T7-TagTM and introduce a C-terminal hexahistidine tag preceded by a thrombin cleavage site. Constructs under the selection of ampicillin were then separately transformed into BL21(DE3) cells co-harboring the pLysS plasmid under the selection of chloramphenicol (Novagen). The authenticity of each construct was verified by sequence analysis of ~300 bp from either end of each cDNA insert within the pET-14b or pET-3a vectors.
For expression of the recombinant mt-GrpE#1 and #2 proteins bearing an N-terminal hexahistidine tag (removable with thrombin), typically 1.5 liters of Luria broth containing 100 µg/ml ampicillin and 40 µg/ml chloramphenicol were inoculated with the transformants and shaken at 30 °C until the A600 nm was equal to 0.5. Synthesis of the recombinant protein was initiated by addition of isopropyl-1-thio-Native PAGE--
Nondenaturing PAGE was performed in 6%
Tris-glycine gels by omitting SDS from the PAGE system (34). The
protein samples were prepared in 25 mM Tris-Cl (pH 8.0), 50 mM NaCl, 50 mM KCl,10 mM MgCl, 10 mM -mecaptoethanol, and 50 units/ml apyrase and
incubated at 4 °C for 6 h prior to electrophoresis at 30 mA for
12 h at 4 °C.
ATPase Assay--
The ATPase activity of recombinant rat
mt-Hsp70 was measured by the conversion of [-32P]ATP
(1 µCi; 4000 Ci/mmol, Bresatec) to
-32Pi
essentially as described previously (8). Assays were carried out for 30 min at 30 °C in 50 µl of reaction mixtures containing 50 mM Tris-Cl (pH 8), 50 mM NaCl, 50 mM KCl, 10 mM MgCl, 2 mM dithiothreitol, 40 µM ATP, and the appropriate proteins.
Reactions were terminated by spotting 2-µl samples onto Polygram CEL
300 polyethyleneimine cellulose plates (Macherey-Nagel) where
[
-32P]ATP was separated from
-32Pi by one-dimensional chromatography
using a solution of 1 M formic acid and 1 M
LiCl. Hydrolyzed ATP was then quantified by Storm PhosphorImager
analysis (Molecular Dynamics). The E. coli DnaJ protein was
purchased from StressGen Biotechnologies Corp. while the recombinant
rat mt-Hsp70, rat mt-GrpE#1 and mouse mt-GrpE#2 proteins were prepared
without their hexahistine tags as described earlier.
RNA Dot Blot Analysis-- For identification of the tissue distribution of the mt-GrpE transcripts, an RNA dot blot with mRNA from 22 different mouse tissues (100-500 ng each) was purchased from CLONTECH (catalog no. 7771-1) and prehybridized, hybridized, and analyzed as described for the Southern blot analysis above.
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RESULTS AND DISCUSSION |
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Identification of Distinct Cytosolic, Microsomal, and Mitochondrial GrpE-like Proteins-- It has previously been established that E. coli GrpE binds strongly to immobilized DnaK in the absence of ATP (20). Utilizing this specific interaction we previously reported the DnaK-affinity purification of a single mitochondrial GrpE homologue (mt-GrpE#1) from rat, porcine, and bovine liver mitochondrial extracts (17). In the present work we again employed DnaK-affinity chromatography, combined with an antiserum to mt-GrpE#1, to identify additional GrpE-like members from a bovine liver organellar extract. Although SDS-PAGE analysis of the protein constituents, that were specifically retained on immobilized DnaK in presence of 1 M KCl but were readily dissociated from DnaK in the presence of 5 mM ATP, revealed only the mt-GrpE#1 protein following staining with Coomassie Brilliant Blue, at least one other related protein appears to be contained in this eluate. Fig. 1A shows the Western blotting pattern observed when the mt-GrpE#1 antiserum was used to probe the protein constituents from the 5 mM ATP eluate (of the DnaK column) and several purified organellar preparations. As suspected, the antiserum detected predominantly mt-GrpE#1 in the 5 mM ATP eluate (Fig. 1A, lane 2) which co-migrated with a single species in the mitochondrial fraction (Fig. 1A, lane 1). The detection of a small amount of a slightly lower molecular weight species was previously determined to be a proteolytic breakdown product of the mt-GrpE#1 protein (Fig. 1A, lane 2) (17).
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Identification of a Second Mammalian mt-GrpE cDNA-- Based on a consensus sequence for the GrpE family (21) a search of data bases revealed a previously uncharacterised mouse EST, from both 13.5-14.5 (GenBankTM accession no. AA049605) and 19.5 (GenBankTM accession no. AA060861) days post conception embryo cDNA libraries. These ESTs exhibit significant homology to our earlier reported rat mt-GrpE#1 cDNA sequence (21) but represent a distinct entity. Complete sequencing of the two EST clones revealed a 1445-bp cDNA (Fig. 2), where GenBankTM accession no. AA049605 (Fig. 2, nucleotides 4-1448) and GenBankTM accession no. AA060861 (Fig. 2, nucleotides 27-1448) were identical in a 1422-bp overlap. By comparison of the predicted amino acid sequence (Fig. 2) with several GrpE homologues (Fig. 3 and Table I), it is concluded that the cDNA encodes a second mouse mitochondrial GrpE protein (named mt-GrpE#2) and in all likelihood only the initiation ATG codon is missing from the coding region of the cDNA clone. In support of this prediction, the presence of an ATG codon on the 5'-end of the cDNA would be complemented by the two adjacent 3'-nucleotides (Fig. 2, numbered 4 and 5) to initiate the translation of a 224-residue putative mitochondrial precursor protein (36). Furthermore, we have previously observed that the initiation ATG codon was absent from a multitude of mt-GrpE#1 cDNAs and corresponding ESTs, and could be recovered only following a 5'-rapid amplification of cDNA ends approach (21). We therefore conclude that some structural feature(s) of the mt-GrpE transcripts may hamper the synthesis of full-length mt-GrpE encoding cDNAs.
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Properties and Interaction of mt-GrpE#1 and #2 with DnaK-- In order to evaluate whether mouse mt-GrpE#2 might function as a co-chaperone for an Hsp70 member, we investigated if mt-GrpE#2 could form a stable complex with DnaK in the absence of ATP, a characteristic of several other GrpE family members (15, 17, 18, 20, 22, 38). The mature portions of both the rat mt-GrpE#1 and mouse mt-GrpE#2 proteins (predicted in Fig. 3) with N-terminal hexahistidine tags were thus synthesized in E. coli and then purified by immobilized metal affinity chromatography (IMAC) (Fig. 5A, lanes 3 and 5, respectively). As seen in Fig. 5, both mt-GrpE#1 and #2 form a specific complex with E. coli DnaK which is stable in the presence of 0.5 M NaCl but readily dissociated in the presence of 5 mM ATP (Fig. 5A, lanes 2 and 4, respectively). Both N-terminal protein sequencing (of the first 10 residues) and mass spectrometric analysis unequivocally revealed the identity of the interacting protein recovered in the 5 mM ATP eluate as E. coli DnaK. Specifically, the predicted mass of mature E. coli DnaK is 68,983.20 Da, while those obtained by mass spectrometry were 68,976 ± 5 Da (Fig. 5A, lane 2) and 68,983 ± 4 Da (Fig. 5A, lane 4). To dismiss the possibility that DnaK might bind directly to the IMAC resin rather than to mt-GrpEs, IMAC purification was also performed on lysates prepared from E. coli not subjected to prior induction of mt-GrpE#1 and #2 synthesis. In this case, DnaK was not detected in the 5 mM ATP eluates (data not shown).
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mt-GrpE#1 and #2 Are Functional Co-chaperones for mt-Hsp70-- The evidence presented thus far would strongly suggest that both mt-GrpE#1 and #2 are authentic GrpE homologues that function as co-chaperones for mammalian Hsp70 members. We therefore investigated whether mt-GrpE#1 and #2 could interact in stable fashion with any of the well known mammalian Hsp70 members (i.e. Hsc70, BiP, and mt-Hsp70, Fig. 6A) in the absence of ATP. Fig. 6B shows that both mt-GrpE#1 and #2 interact specifically with mt-Hsp70 during native PAGE analysis. This interaction is not observed with cytosolic/nuclear Hsc70 nor ER BiP (Fig. 6B). Such specificity in the interaction between an Hsp70 member and its GrpE partner is not uncommon, as E. coli GrpE neither interacts with or is capable of stimulating the ATPase activity of the cytosolic S. cerevisiae Hsp70 member (Ssa1p) (41). Of further interest here is the observation that mt-Hsp70 oligomers are apparently dissociated by mt-GrpE#1 and #2 to permit the formation of a stable heterologous complex (Fig. 6B). An apparent DnaK oligomer dissociating activity has been ascribed to E. coli GrpE previously (12). In that case it is suggested dimeric GrpE serves to stabilize monomeric DnaK in a 2:1 complex rather than actively disrupting DnaK oligomers. In the ER, it has been suggested that BiP interconversion between the oligomeric (inactive) and monomeric (active) state allows the cell to maintain a constant level of functionally active BiP available (42). Thus GrpE molecules, in addition to their role as nucleotide exchange factors for the stimulation of Hsp70 ATPase activity, may also serve to maintain adequate cellular levels of their functionally active Hsp70 counterpart by modulating the interconversion of oligomeric (inactive) and monomeric (active) forms of these chaperones.
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Expression of mt-GrpE#2-- A ubiquitous but varying level of expression for the rat mt-GrpE#1 transcript (~1.2 kilobase pairs) was previously seen in eight different tissues (21). Using a more expansive dot blot of murine mRNAs obtained from 22 different sources (Fig. 7A) and normalized to the mRNA expression levels of eight different housekeeping genes, the expression of both the mt-GrpE#2 and #1 transcripts appeared to be ubiquitous (Fig. 7, B and C, respectively). We rule out nonspecific hybridization as negative controls such as: yeast total RNA and tRNA (grid position = F1 and F2, respectively); E. coli rRNA and DNA (grid position = F3 and F4, respectively); synthetic poly(rA) (grid position = G1); and Cot1 DNA (representing the most abundant repetitive sequences, grid position = G2) did not give rise to any hybridization signal. Furthermore, since the stringency of the hybridization was identical to that seen for the Southern blot in Fig. 3, cross-hybridization of the mt-GrpE#2 probe with mt-GrpE#1 transcript is most unlikely. Despite this apparent ubiquitous expression of the mt-GrpE#2 transcript, we never detected any proteins in the 5 mM ATP eluates (of the DnaK columns) with an N-terminal sequence related to mt-GrpE#2. Neither did antibodies raised against the 5 mM ATP eluates recognize recombinant mt-GrpE#2, while recombinant mt-GrpE#1 was readily detected. The reason for this apparent contradiction between the ubiquitous presence of mt-GrpE#2 transcript and the absence of the corresponding translation product is not clear, but a similar situation has been documented for an osmotin-like protein in plants (45).
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ACKNOWLEDGEMENTS |
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We thank Joan Hoogenraad for assistance with the tissue culture work, Yoji Hayasaka for mass spectrometric analysis, and Helen Healey for help with the design of expression vectors.
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FOOTNOTES |
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* This work was supported by grants from the National Health and Medical Research Council and from the Australian Research Council (to N. J. H. and P. B. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF041060.
¶ To whom correspondence should be addressed. Tel.: 61 8 8303 6670; Fax: 61 8 8303 7116, E-mail: dnaylor{at}waite.adelaide.edu.au.
Recipient of a Cooperative Research Centre for Viticulture
Scholarship.
The abbreviations used are: Hsp70, 70 kDa heat shock protein; EB, equilibration buffer; ER, endoplasmic reticulum; Hap, Hsp70/Hsc70-associating protein; Hsc70, the constitutive isoform of Hsp70; IMAC, immobilized metal affinity chromatography; mt, mitochondrial; EST, expressed sequence tag; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinebp, base pair(s).
2 D. J. Naylor, N. J. Hoogenraad, and P. B. Høj, manuscript in preparation.
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REFERENCES |
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