The NH2-terminal Domain of the Chloroplast GrpE Homolog CGE1 Is Required for Dimerization and Cochaperone Function in Vivo
2007; Elsevier BV; Volume: 282; Issue: 15 Linguagem: Inglês
10.1074/jbc.m608854200
ISSN1083-351X
AutoresFelix Willmund, Timo Mühlhaus, Maria Wojciechowska, Michael Schroda,
Tópico(s)Heat shock proteins research
ResumoGrpE proteins function as nucleotide exchange factors for DnaK-type Hsp70s. We have previously identified a chloroplast homolog of GrpE in Chlamydomonas reinhardtii, termed CGE1. CGE1 exists as two isoforms, CGE1a and CGE1b, which are generated by temperature-dependent alternative splicing. CGE1b contains additional valine and glutamine residues in its extreme NH2-terminal region. Here we show that CGE1a is predominant at lower temperatures but that CGE1b becomes as abundant as CGE1a at elevated temperatures. Coimmunoprecipitation experiments revealed that CGE1b had a ∼25% higher affinity for its chloroplast chaperone partner HSP70B than CGE1a. Modeling of the structure of CGE1b revealed that the extended α-helix formed by GrpE NH2 termini is 34 amino acids longer in CGE1 than in Escherichia coli GrpE and appears to contain a coiled coil motif. Progressive deletions of this coiled coil increasingly impaired the ability of CGE1 to form dimers, to interact with DnaK at elevated temperatures, and to complement temperature-sensitive growth of a ΔgrpE E. coli strain. In contrast, deletion of the four-helix bundle required for dimerization of E. coli GrpE did not affect CGE1 dimer formation. Circular dichroism measurements revealed that CGE1, like GrpE, undergoes two thermal transitions, the first of which is in the physiologically relevant temperature range (midpoint ∼45 °C). Truncating the NH2-terminal coiled coil shifted the second transition to lower temperatures, whereas removal of the four-helix bundle abolished the first transition. Our data suggest that bacterial GrpE and chloroplast CGE1 share similar structural and biochemical properties, but some of these, like dimerization, are realized by different domains. GrpE proteins function as nucleotide exchange factors for DnaK-type Hsp70s. We have previously identified a chloroplast homolog of GrpE in Chlamydomonas reinhardtii, termed CGE1. CGE1 exists as two isoforms, CGE1a and CGE1b, which are generated by temperature-dependent alternative splicing. CGE1b contains additional valine and glutamine residues in its extreme NH2-terminal region. Here we show that CGE1a is predominant at lower temperatures but that CGE1b becomes as abundant as CGE1a at elevated temperatures. Coimmunoprecipitation experiments revealed that CGE1b had a ∼25% higher affinity for its chloroplast chaperone partner HSP70B than CGE1a. Modeling of the structure of CGE1b revealed that the extended α-helix formed by GrpE NH2 termini is 34 amino acids longer in CGE1 than in Escherichia coli GrpE and appears to contain a coiled coil motif. Progressive deletions of this coiled coil increasingly impaired the ability of CGE1 to form dimers, to interact with DnaK at elevated temperatures, and to complement temperature-sensitive growth of a ΔgrpE E. coli strain. In contrast, deletion of the four-helix bundle required for dimerization of E. coli GrpE did not affect CGE1 dimer formation. Circular dichroism measurements revealed that CGE1, like GrpE, undergoes two thermal transitions, the first of which is in the physiologically relevant temperature range (midpoint ∼45 °C). Truncating the NH2-terminal coiled coil shifted the second transition to lower temperatures, whereas removal of the four-helix bundle abolished the first transition. Our data suggest that bacterial GrpE and chloroplast CGE1 share similar structural and biochemical properties, but some of these, like dimerization, are realized by different domains. Molecular chaperones of the Hsp70 family essentially consist of two domains, an NH2-terminal ATPase domain and a COOH-terminal substrate-binding domain. In the ATP-bound state, Hsp70s have a low affinity for substrates; in the ADP-bound state, affinity for substrates is high (for reviews, see Refs. 1Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2435) Google Scholar and 2Mayer M.P. Bukau B. Cell. Mol. Life Sci. 2005; 62: 670-684Crossref PubMed Scopus (2091) Google Scholar). The conversion between both states is regulated by cochaperones; J-domain proteins, which also supply Hsp70s with specific substrates, normally trigger ATP hydrolysis and thus catalyze the conversion of Hsp70s into the ADP-bound state with high affinity for substrate (3Liberek K. Marszalek J. Ang D. Georgopoulos C Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Crossref PubMed Scopus (693) Google Scholar, 4Kelley W.L. Trends Biochem. Sci. 1998; 23: 222-227Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Nucleotide exchange factors, like Bag1 for eukaryotic-type Hsp70s or GrpE for bacterial Hsp70s of the DnaK type, mediate the exchange of ADP by ATP and thus catalyze the reconversion of Hsp70 from the ADP-bound to the ATP-bound state with low affinity for substrate (3Liberek K. Marszalek J. Ang D. Georgopoulos C Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Crossref PubMed Scopus (693) Google Scholar, 5Gässler C.S. Wiederkehr T. Brehmer D. Bukau B. Mayer M.P. J. Biol. Chem. 2001; 276: 32538-32544Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). GrpE-type nucleotide exchange factors are required only by DnaK-type Hsp70s, which contain a conserved region within their ATPase domain, allowing them to bind nucleotides with particularly high affinity and to hydrolyze ATP at high rates (6Brehmer D. Rüdiger S. Gässler C.S. Klostermeier D. Packschies L. Reinstein J. Bukau B. Nat. Struct. Biol. 2001; 8: 427-432Crossref PubMed Scopus (190) Google Scholar). Such DnaK-type Hsp70s exist not only in bacteria but also in mitochondria and chloroplasts, where their GrpE-type cochaperones are termed MGE1 (7Laloraya S. Gambill B.D. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6481-6485Crossref PubMed Scopus (140) Google Scholar) and CGE1 (8Schroda M. Vallon O. Whitelegge J.P. Beck C.F. Wollman F.-A. Plant Cell. 2001; 13: 2823-2839Crossref PubMed Scopus (94) Google Scholar), respectively. The Hsp70 partner of CGE1 in the chloroplast of Chlamydomonas is called HSP70B (9Drzymalla C. Schroda M. Beck C.F. Plant Mol. Biol. 1996; 31: 1185-1194Crossref PubMed Scopus (57) Google Scholar, 10Schroda M. Photosynth. Res. 2004; 82: 221-240Crossref PubMed Scopus (116) Google Scholar). To catalyze nucleotide exchange, GrpEs interact with their Hsp70 partners as dimers (8Schroda M. Vallon O. Whitelegge J.P. Beck C.F. Wollman F.-A. Plant Cell. 2001; 13: 2823-2839Crossref PubMed Scopus (94) Google Scholar, 11Schönfeld H.-J. Schmidt D. Schröder H. Bukau B. J. Biol. Chem. 1995; 270: 2183-2189Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 12Deloche O. Georgopoulos C. J. Biol. Chem. 1996; 271: 23960-23966Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). According to the crystal structure of Escherichia coli GrpE in complex with the ATPase domain of DnaK, it is only one GrpE molecule of the dimer that contacts DnaK (13Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.-U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar). E. coli GrpE consists of four domains (Refs. 13Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.-U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar and 14Harrison C. Cell Stress Chaperones. 2003; 8: 218-224Crossref PubMed Scopus (97) Google Scholar; see also Fig. 4), which in the following are listed from the COOH to the NH2 termini: (i) the β-sheet domain, which contributes most contacts with the DnaK ATPase domain (by introducing a conformational change in the DnaK nucleotide-binding cleft, it mediates nucleotide exchange); (ii) the four-helix bundle, to which each GrpE monomer contributes two short α-helices and which serves as a dimerization platform (15Gelinas A.D. Langsetmo K. Toth J. Bethoney K.A. Stafford W.F. Harrison C.J. J. Mol. Biol. 2002; 323: 131-142Crossref PubMed Scopus (35) Google Scholar, 16Mehl A.F. Heskett L.D. Jain S.S. Demeler B. Protein Sci. 2003; 12: 1205-1215Crossref PubMed Scopus (10) Google Scholar); (iii) the extended, paired α-helices, which require the four-helix bundle for pairing (15Gelinas A.D. Langsetmo K. Toth J. Bethoney K.A. Stafford W.F. Harrison C.J. J. Mol. Biol. 2002; 323: 131-142Crossref PubMed Scopus (35) Google Scholar, 16Mehl A.F. Heskett L.D. Jain S.S. Demeler B. Protein Sci. 2003; 12: 1205-1215Crossref PubMed Scopus (10) Google Scholar) and which appear to serve as a thermosensor (local melting of the paired helix at heat shock temperatures drastically lowers the efficiency of GrpE to catalyze nucleotide exchange in DnaK (15Gelinas A.D. Langsetmo K. Toth J. Bethoney K.A. Stafford W.F. Harrison C.J. J. Mol. Biol. 2002; 323: 131-142Crossref PubMed Scopus (35) Google Scholar, 17Grimshaw J.P.A. Jelesarov I. Schönfeld H.-J. Christen P. J. Biol. Chem. 2001; 276: 6098-6104Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 18Grimshaw J.P. Jelesarov I. Siegenthaler R.K. Christen P. J. Biol. Chem. 2003; 278: 19048-19053Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar)); and (iv) the unstructured NH2 terminus, which may interact with the substrate-binding domain of DnaK (19Brehmer D. Gassler C. Rist W. Mayer M.P. Bukau B. J. Biol. Chem. 2004; 279: 27957-27964Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 20Chesnokova L.S. Slepenkov S.V. Protasevich I.I. Sehorn M.G. Brouillette C.G. Witt S.N. Biochemistry. 2003; 42: 9028-9040Crossref PubMed Scopus (27) Google Scholar). We have reported previously that by a temperature-dependent alternative splicing process, the gene encoding the chloroplast GrpE homolog CGE1 gives rise to two transcripts termed CGE1a and CGE1b (8Schroda M. Vallon O. Whitelegge J.P. Beck C.F. Wollman F.-A. Plant Cell. 2001; 13: 2823-2839Crossref PubMed Scopus (94) Google Scholar). The CGE1b transcript contains six additional nucleotides coding for valine and glutamine, which are the fourth and fifth NH2-terminal residues of mature CGE1b. In this study, we demonstrate that the CGE1 NH2-terminal region strongly affects the biochemical properties of the protein; first, valine-glutamine in CGE1b increased its affinity for HSP70B, and second, dimerization and functionality of CGE1 in vivo required a coiled coil domain situated at the NH2 terminus of the protein. Strains and Culture Conditions—Chlamydomonas reinhardtii was grown mixotrophically in TAP medium (21Harris E.H. The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, Inc., San Diego, CA1989: 26-27Google Scholar) on a rotatory shaker at 25 °C and ∼30 microeinsteins m-2 s-1. Wild type strain 137c (mt-) was used for the experiments of Fig. 1, and cw15 strain CF185 (22Schroda M. Vallon O. Wollman F.-A. Beck C.F. Plant Cell. 1999; 11: 1165-1178Crossref PubMed Scopus (245) Google Scholar) was used for the isolation of HSP70B and the experiments of Fig. 3, C and D.FIGURE 3Binding competition assays and analysis of CGE1a/b dimers. A, HSP70B isolated from Chlamydomonas (7 nm) was mixed with CGE1a and CGE1b (42 nm each) in the absence of ATP and incubated for 30 min. After 10% of the reaction mixture had been removed as input control, HSP70B was immunoprecipitated, and precipitating proteins were separated on an SDS-14% polyacrylamide gel and analyzed by immunoblotting. Incubations and immunoprecipitations were entirely carried out at 23 or 37 °C, respectively. B, the experiment was performed as that in A at 23 °C, but the ratio of CGE1a to CGE1b proteins that were mixed with HSP70B was increased as indicated. C, Chlamydomonas cells were grown for 16 h at 37 °C or incubated at 41 °C for 4 h. Cells were depleted from ATP, and soluble proteins were isolated. After removing 1% as input control, HSP70B was immunoprecipitated, and precipitating proteins (IP) were separated on an SDS-14% polyacrylamide gel and analyzed by immunoblotting. D, ATP-depleted soluble cell extracts from Chlamydomonas cells that were grown at 23 °C or incubated at 41°C for 2 h (upper panel), or 40 ng of purified CGE1a (a), CGE1b (b), or a mixture of both (a + b) (lower panel) were separated on native 8-14% polyacrylamide gels and analyzed by immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Polyacrylamide Electrophoreses and Gel Blot Analyses—SDS-PAGE and gel blot analyses were performed as described earlier (23Liu C. Willmund F. Whitelegge J.P. Hawat S. Knapp B. Lodha M. Schroda M. Mol. Biol. Cell. 2005; 16: 1165-1177Crossref PubMed Scopus (103) Google Scholar). Native PAGE was carried out according to Schägger et al. (24Schägger H. Cramer W.A. von Jagow G. Anal. Biochem. 1994; 217: 220-230Crossref PubMed Scopus (1043) Google Scholar). Antisera used were against HSP70B (22Schroda M. Vallon O. Wollman F.-A. Beck C.F. Plant Cell. 1999; 11: 1165-1178Crossref PubMed Scopus (245) Google Scholar), CF1α (25Lemaire C. Wollman F.A. J. Biol. Chem. 1989; 264: 10235-10242Abstract Full Text PDF PubMed Google Scholar), and CGE1 (8Schroda M. Vallon O. Whitelegge J.P. Beck C.F. Wollman F.-A. Plant Cell. 2001; 13: 2823-2839Crossref PubMed Scopus (94) Google Scholar). Detections were done with ECL. Cloning, Expression, and Purification of CGE1 Derivatives, DnaK, and HSP70B—The coding regions of CGE1b, Δ9, Δ16, Δ25, Δ45, and Δ71, were amplified by PCR from cDNA clone AV391963 (encoding CGE1b) with 5′ primers 5′-GGCTCGTCGTTTTGGCTCTTCGAACGCGGCTG-3′,5′-GGACTAGTGCTCTTCCAACGCCCCTGCTGAGGAGGCGGC-3′,5′-GGACTAGTGCTCTTCCAACGCTACCCCCCTGGAGCGTGC-3′, 5′-GGACTAGTGCTCTTCCAACGCTCTGGACAGCGAGACC-3′,5′-CTTGGCTCTTCGAACGCTGAGATGGGTCGC-3′,5′-GGACTAGTGCTCTTCCAACGCCAAGGACCAGTACC-3′, and 3′ primer T7. For ΔHB, primers 5′-GGCTCGTCGTTTTGGCTCTTCGAACGCGGCTG-3′ and 5′-CTTCTCGAGTTAGCCGCGCACGCTGTCGGTGA-3′ were used. For CGE1a, primers 5′-GGCTCGTCGTTTTGGCTCTTCGAACGCGGCTG-3′ and T7 were used with pMS213 (cDNA clone encoding CGE1a) as a template (8Schroda M. Vallon O. Whitelegge J.P. Beck C.F. Wollman F.-A. Plant Cell. 2001; 13: 2823-2839Crossref PubMed Scopus (94) Google Scholar). PCR products were digested with SapI and XhoI and cloned into SapI-XhoI-digested pTYB11 (NEB, Frankfurt, Germany), giving pMS300 (1a), pMS301 (1b), pMS365 (Δ9), pMS367 (Δ16), pMS369 (Δ25), pMS295 (Δ45), pMS412 (Δ71), and pMS413 (ΔHB). Correct cloning was verified by sequencing. CGE1 derivatives were expressed as fusion proteins in E. coli ER2566 and purified by chitin affinity chromatography according to the manufacturer's instructions (NEB) but including a wash step with 5 mm ATP. Pure proteins were dialyzed three times against 2 liters of KH buffer (20 mm Hepes-KOH, pH 7.2, 80 mm KCl) or 20 mm sodium phosphate buffer, pH 7.5, concentrated in Amicon Ultra-4 tubes (Millipore, Molsheim, France), frozen in liquid nitrogen, and stored at -80 °C. HSP70B (BEc) also was isolated by chitin affinity chromatography as described previously (26Willmund F. Schroda M. Plant Physiol. 2005; 138: 2310-2322Crossref PubMed Scopus (60) Google Scholar), but the protein expressed here did not contain a hexahistidine tag. HSP70B (BCr) and DnaK were isolated by CGE1 affinity chromatography as described previously (23Liu C. Willmund F. Whitelegge J.P. Hawat S. Knapp B. Lodha M. Schroda M. Mol. Biol. Cell. 2005; 16: 1165-1177Crossref PubMed Scopus (103) Google Scholar, 27Liu C. Willmund F. Golecki J.R. Cacace S. Hess B. Markert C. Schroda M. Plant J. 2007; (in press)Google Scholar). The purity of the BCr preparation was checked by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of tryptically digested BCr protein as described previously (28Schmidt M. Gessner G. Luff M. Heiland I. Wagner V. Kaminski M. Geimer S. Eitzinger N. Reissenweber T. Voytsekh O. Fiedler M. Mittag M. Kreimer G. Plant Cell. 2006; 18: 1908-1930Crossref PubMed Scopus (149) Google Scholar). Of the 91 peptides identified for Chlamydomonas HSP70s, 95% were from HSP70B, and 1-2%, respectively, were from endoplasmic reticulum-resident BIP1/2, cytosolic HSP70A, and mitochondrial HSP70C. The construction of vectors for the expression of CGE1 derivatives in strain OD212 was as follows. The CGE1b coding region was amplified by PCR from pMS205 (8Schroda M. Vallon O. Whitelegge J.P. Beck C.F. Wollman F.-A. Plant Cell. 2001; 13: 2823-2839Crossref PubMed Scopus (94) Google Scholar) (contains the CGE1b cDNA with coding regions for NH2- and COOH-terminal hexahistidine tags in expression vector pQE-9 (Qiagen, Hilden, Germany)) with primers 5′-CAGAATTCATTAAAGAGGAGAAATTAACTATGTACGTATCGCATC-3′ and 5′-CCCAAGCTTAGTGATGGTGATGGTGATGGTGATGGTGATGGTAACC-3′. The 777-bp PCR product was digested with EcoRI and HindIII and ligated into EcoRI-HindIII-digested pQE-9, giving pMS397. Next, the coding regions of the CGE1 derivatives were PCR-amplified with primers 5′-GGATCCCAGGTTTACGTACAGAAC-3′ and 5′-TTGGGTAACCCTCCTCAGAGCTAGCCGCAGGGCCGT-3′ using pMS300, pMS301, pMS367, pMS369, and pMS295 as templates. PCR products were then digested with SnaBI and BstEII and ligated into SnaBI-BstEII-digested pMS397, generating pMS399 (1a), pMS400 (1b), pMS401 (Δ16), pMS402 (Δ25), and pMS398 (Δ45). To remove regions coding for COOH-terminal histidine tags, the latter constructs were digested with BstXI and BlpI, and a ∼1-kb BstXI-BlpI fragment from BstXI-BlpI-digested pMS301 was inserted, giving pMS404 (1a), pMS405 (1b), pMS406 (Δ16), pMS407 (Δ25), and pMS403 (Δ45). pMS408 (Δ9) was constructed by ligating a ∼500-bp PCR product (using primers 5′-CAGAATTCATTAAAGAGGAGAAATTAACTATGTACGTATCGCATC-3′ and 5′-CCCAAGCTTAGTGATGGTGATGGTGATGGTGATGGTGATGGTAACC-3′ on pMS365) digested with SnaBI and BstXI into SnaBI-BstXI-digested pMS404. Correct cloning was verified by sequencing. Glutaraldehyde Cross-links—Proteins were incubated in KMH buffer (20 mm Hepes-KOH, pH 7.2, 80 mm KCl, and 2.5 mm MgCl2) for 10-30 min. Then glutaraldehyde (0.05-0.1%) was added, and the incubation was continued for another 10-20 min. Cross-linking was stopped by the addition of one volume of 2× Laemmli buffer (125 mm Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 10% β-mercaptoethanol, 0.005% bromphenol blue) containing 400 mm glycine. Immunoprecipitations and Native Dimer Assays—For affinity assays, 20-μl reaction mixtures containing 50 ng of HSP70B, 15 μg of bovine serum albumin, and 0.75 units of apyrase in KMH buffer were incubated at 23 °C for 15 min. Mixtures were then diluted to 100 μl with KMH buffer containing 100 ng of CGE1b, CGE1a as indicated, and 250 μg of bovine serum albumin. After further incubation for 30 min, 200 μl of KMH and 40 μl of Protein A-Sepharose beads coupled with 20 μl of polyclonal anti-HSP70B serum (as described in Ref. 8Schroda M. Vallon O. Whitelegge J.P. Beck C.F. Wollman F.-A. Plant Cell. 2001; 13: 2823-2839Crossref PubMed Scopus (94) Google Scholar) were added. After mixing for 1 h on an overhead shaker, beads were washed four times with KMH and twice with 10 mm Tris-HCl, pH 7, and proteins were eluted by boiling for 45 s after the addition of one volume of 2× Laemmli buffer. For immunoprecipitations from cell extracts, Chlamydomonas cells from a 300-ml culture with a density of ∼2 × 106 cells/ml had been grown overnight at 37 °C or subjected to heat shock at 41 °C for 4 h. Cells were harvested and lysed by sonication on ice in 4 ml of lysis buffer (20 mm Hepes, pH 7.2, 1 mm MgCl2, 20 mm KCl, 150 mm NaCl, 0.25× protease inhibitor mixture (Roche Applied Science), 10 μm carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and 5 units of apyrase). Lysates were loaded onto a sucrose cushion (20 mm Hepes-KOH, pH 7.2, 0.6 m sucrose) and centrifuged in a TI50 rotor for 30 min at 152,000 × g and 4 °C. 2 ml of the supernatant were incubated with 100 μl of Protein A-Sepharose beads coupled with 200 μl of polyclonal anti-HSP70B serum. After mixing for 1 h on an overhead shaker, beads were washed four times with lysis buffer and twice with 10 mm Tris-HCl, pH 7, and proteins were eluted by boiling after the addition of one volume of 2× Laemmli buffer. Quantification of CGE1a/b bands was done from scanned ECL films using the QuantityOne-4.5.1 program (Bio-Rad). For dimerization assays, 1 μm CGE1a, 1 μm CGE1b, or a 0.5 μm concentration of both was incubated in 25 mm Hepes-KOH, pH 8, 50 mm KCl, 10 mm β-mercaptoethanol, 0.1 mm EDTA, 2.5 mm MgCl2, 20% glycerol for 30 min at 23 °C. The reaction mixture was then diluted 10-fold in 50 mm BisTris 2The abbreviation used is: BisTris, bis-(2-hydroxyethyl)iminotris(hydroxymethyl)methane. -HCl, pH 7.0, 0.5 m ϵ-aminocaproic acid, 15% glycerol, 1 mm MgCl2, 10 mm KCl, 0.004% Ponceau-S, and 10% of the mix was loaded onto a native gel. Homology Modeling of CGE1b—As a template for homology modeling, the structure of the E. coli GrpE dimer bound to its HSP70 chaperone partner DnaK was used (13Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.-U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar) (Protein Data Bank entry 1DKG, chains A and B). Pairwise alignment of CGE1b and GrpE was done based on a multiple alignment of CGE1 with GrpE homologs of bacterial and mitochondrial origins (8Schroda M. Vallon O. Whitelegge J.P. Beck C.F. Wollman F.-A. Plant Cell. 2001; 13: 2823-2839Crossref PubMed Scopus (94) Google Scholar). Secondary structure prediction of the NH2-terminal region of CGE1, which is not conserved between CGE1 and GrpE, was done with the programs JPRED, PHD, PROF, PSIpred, PSSP, and SSpro accessed via the Columbia metamask (available on the World Wide Web at cubic.bioc.columbia.edu/predictprotein/submit_meta.html). Tropomyosin (29Whitby F.G. Phillips Jr., G.N. Proteins. 2000; 38: 49-59Crossref PubMed Scopus (158) Google Scholar) (Protein Data Bank entry 1C1G, amino acids 70-122 of chain A and 355-407 of chain B) as an appropriate template for the modeling of the CGE1 NH2-terminal region was found by using the hydrophobic pattern identified in this region as a query for BLAST-P. Pairwise alignment of CGE1b and Tropomyosin was done on the basis of similar hydrophobicity. 20 series of 100 models each with plausible alternative alignments (including an α-helical restraint for Ala1-Ala12 and Ala222-Ala233, respectively, based on secondary structure prediction) was generated with the MODELLER 7v06 software within InsightII (Accelrys, San Diego, CA). The five models with highest ranking (based on probability density function and energy values) and best root mean square values as determined by ProFit (A. C. R. Martin; available on the World Wide Web at www.bioinf.org.uk/software/profit/) were tested with the Procheck version 3.5.4 program (30Morris A.L. MacArthur M.W. Hutchinson E.G. Thornton J.M. Proteins. 1992; 12: 345-364Crossref PubMed Scopus (1420) Google Scholar) and checked manually. The model chosen was then energy-minimized three times using the CHARMM force field in InsightII. During the first minimization (600 steps and 0.001 final convergence) the backbone and the region between Arg24-Glu221 and Ala295-Glu442 were fixed. During the second minimization (again 600 steps and 0.001 final convergence), only the region Arg24-Glu221 and Ala295-Glu442 was fixed. The entire model was fixed during the final minimization (30 steps). Circular Dichroism Measurements—Circular dichroism was measured with a Jasco J-810 spectropolarimeter (Jasco, Tokyo, Japan) using a thermostated cuvette with a 1-mm path length. Temperature was controlled with a programmable water bath. At fixed temperatures (25 °C), three spectra between 300 and 190 nm (bandwidth 1 nm) were recorded every 0.1 nm at a scan speed of 200 nm min-1 and averaged. For these experiments, proteins had been dialyzed against 20 mm sodium phosphate, pH 7.5, and proteins were used at a concentration of 10 μm. Time courses of temperature-induced conformational changes were followed by continuously monitoring the ellipticity at 222 nm (bandwidth 1 nm). The cuvette was heated by 0.5 °C min-1, and measurements were taken once every 2 min. For these experiments, proteins had been dialyzed against KH buffer, and proteins were used at 20 μm concentration. Temperature-dependent Alternative Splicing of CGE1 Transcripts Leads to the Differential Accumulation of CGE1a and CGE1b Isoforms—The recently available Chlamydomonas genome sequence (available on the World Wide Web at genome.jgi-psf.org/Chlre3/Chlre3.home.html) allowed us to elucidate that the CGE1 gene consists of eight exons and seven introns and that it is the first CGE1 intron that is alternatively spliced (Fig. 1A). Alternative splicing was shown to be temperature-dependent (i.e. CGE1a represented 60-80% of the CGE1 message at 25 °C but declined to ∼30% in favor of CGE1b after a 40-min heat shock) (8Schroda M. Vallon O. Whitelegge J.P. Beck C.F. Wollman F.-A. Plant Cell. 2001; 13: 2823-2839Crossref PubMed Scopus (94) Google Scholar). To test whether temperature-dependent changes in CGE1 message composition also were reflected at the protein level, we subjected Chlamydomonas cells to different temperature treatments and analyzed the cellular accumulation of CGE1a and CGE1b protein. As shown in Fig. 1B, CGE1a was much more abundant than CGE1b at 15 and 25 °C. However, CGE1b levels increased slightly at 30 °C, and CGE1b became equally abundant as CGE1a at 37 and 41 °C. We were surprised to see that we could separate CGE1a and CGE1b by SDS-PAGE, since the two proteins differ only by 227.3 Da, the mass of valine and glutamine. To verify that the CGE1 double band observed in Fig. 1B originated from the CGE1a/b isoforms, we compared the migration pattern of CGE1 from heat-shocked Chlamydomonas cells with that of purified CGE1a/b proteins that had been heterologously expressed in E. coli. The masses of heterologously expressed CGE1a and CGE1b determined by mass spectrometry were 23812.0 and 24040.0, respectively, which matched the masses calculated from the amino acid sequences (23812.19 and 24039.46, respectively). As presented in Fig. 1C, also purified CGE1a and CGE1b could be separated in SDS-polyacrylamide gels, and they co-migrated exactly with the two isoforms from Chlamydomonas cells. We conclude that the temperature-dependent alternative splicing of the CGE1 transcript leads to a temperature-dependent accumulation of CGE1a and CGE1b proteins. HSP70B Purified from E. coli Does Not Interact with CGE1, but HSP70B Purified from Chlamydomonas Does—According to current knowledge, GrpE-type proteins do not have any enzyme activities by themselves, but they act as cochaperones for Hsp70s (14Harrison C. Cell Stress Chaperones. 2003; 8: 218-224Crossref PubMed Scopus (97) Google Scholar). Hence, if the differential accumulation of the CGE1a/b isoforms was of any biological significance, it was expected to be by interaction with HSP70B, the chloroplast Hsp70 partner of CGE1 (8Schroda M. Vallon O. Whitelegge J.P. Beck C.F. Wollman F.-A. Plant Cell. 2001; 13: 2823-2839Crossref PubMed Scopus (94) Google Scholar). Since we intended to study possible effects of CGE1a/b on HSP70B in vitro, we first purified HSP70B. This we did from E. coli cells that heterologously expressed HSP70B (BEc) and from Chlamydomonas cell extracts (BCr; Fig. 2A) (23Liu C. Willmund F. Whitelegge J.P. Hawat S. Knapp B. Lodha M. Schroda M. Mol. Biol. Cell. 2005; 16: 1165-1177Crossref PubMed Scopus (103) Google Scholar, 26Willmund F. Schroda M. Plant Physiol. 2005; 138: 2310-2322Crossref PubMed Scopus (60) Google Scholar). Next, we used glutaraldehyde cross-linking to test whether our HSP70B preparations were capable of interacting with CGE1 in vitro. As shown in Fig. 2B, most of purified CGE1a existed as dimers, as expected from previous observations with native gels (8Schroda M. Vallon O. Whitelegge J.P. Beck C.F. Wollman F.-A. Plant Cell. 2001; 13: 2823-2839Crossref PubMed Scopus (94) Google Scholar). In the absence of ATP, CGE1a readily formed a complex with BCr that was disrupted by ATP. In contrast, CGE1a did not interact with BEc. Interestingly, BEc and BCr cross-linked in the absence of CGE1 already displayed different migration properties; whereas BEc migrated as oligomers, dimers, and as a compact species of ∼50 kDa, BCr appeared not to form oligomers or dimers but mainly occurred as the compact ∼50-kDa species and as a less compact ∼70-kDa species. Since the latter vanished entirely upon the addition of CGE1a in the absence of ATP but reappeared upon the addition of ATP, it appears to be this less compact ∼70-kDa species of HSP70B that is able to interact with CGE1. In summary, recombinant CGE1a efficiently formed dimers and formed ATP-sensitive complexes with HSP70B isolated from Chlamydomonas but not with recombinant HSP70B from E. coli. Apparently, recombinant HSP70B stably assumed nonfunctional conformations. CGE1b in Vitro and in Vivo Has a Higher Affinity for HSP70B than CGE1a—We suggested previously that the CGE1a/b isoforms may differ in their affinity for HSP70B (8Schroda M. Vallon O. Whitelegge J.P. Beck C.F. Wollman F.-A. Plant Cell. 2001; 13: 2823-2839Crossref PubMed Scopus (94) Google Scholar). To test this idea, we used HSP70B isolated from Chlamydomonas, mixed it with equal amounts of CGE1a and CGE1b in the absence of ATP, immunoprecipitated HSP70B from the mixture, and assayed how much CGE1a/b coprecipitated with HSP70B. Although crude, the advantage of this assay was that it required only small amounts of the precious BCr. The assay was performed at 23 °C and at 37 °C to monitor possible temperature effects. As demonstrated in Fig. 3A, little but reproducibly more CGE1b than CGE1a coprecipitated with HSP70B at 23 °C. At 37 °C, the affinity of both CGE1a and CGE1b for HSP70B was dramatically reduced compared with 23 °C, but also at 37 °C CGE1b had a higher affinity for HSP70
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