Interaction between the Nucleotide Exchange Factor Mge1 and the Mitochondrial Hsp70 Ssc1
1999; Elsevier BV; Volume: 274; Issue: 16 Linguagem: Inglês
10.1074/jbc.274.16.11275
ISSN1083-351X
AutoresSayuri Sakuragi, Qinglian Liu, Elizabeth A. Craig,
Tópico(s)Protein Structure and Dynamics
ResumoFunction of Hsp70s such as DnaK of theEscherichia coli cytoplasm and Ssc1 of the mitochondrial matrix of Saccharomyces cerevisiae requires the nucleotide release factors, GrpE and Mge1, respectively. A loop, which protrudes from domain IA of the DnaK ATPase domain, is one of six sites of interaction revealed in the GrpE:DnaK co-crystal structure and has been implicated as a functionally important site in both DnaK and Ssc1. Alanine substitutions for the amino acids (Lys-108 and Arg-213 of Mge1) predicted to interact with the Hsp70 loop were analyzed. Mge1 having both substitutions was able to support growth in the absence of the essential wild-type protein. K108A/R213A Mge1 was able to stimulate nucleotide release from Ssc1 and function in refolding of denatured luciferase, albeit higher concentrations of mutant protein than wild-type protein were required. In vitro and in vivo assays using K108A/R213A Mge1 and Ssc1 indicated that the disruption of contact at this site destabilized the interaction between the two proteins. We propose that the direct interaction between the loop of Ssc1 and Mge1 is not required to effect nucleotide release but plays a role in stabilization of the Mge1-Ssc1 interaction. The robust growth of the K108A/R213A MGE1 mutant suggests that the interaction between Mge1 and Ssc1 is tighter than required for functionin vivo. Function of Hsp70s such as DnaK of theEscherichia coli cytoplasm and Ssc1 of the mitochondrial matrix of Saccharomyces cerevisiae requires the nucleotide release factors, GrpE and Mge1, respectively. A loop, which protrudes from domain IA of the DnaK ATPase domain, is one of six sites of interaction revealed in the GrpE:DnaK co-crystal structure and has been implicated as a functionally important site in both DnaK and Ssc1. Alanine substitutions for the amino acids (Lys-108 and Arg-213 of Mge1) predicted to interact with the Hsp70 loop were analyzed. Mge1 having both substitutions was able to support growth in the absence of the essential wild-type protein. K108A/R213A Mge1 was able to stimulate nucleotide release from Ssc1 and function in refolding of denatured luciferase, albeit higher concentrations of mutant protein than wild-type protein were required. In vitro and in vivo assays using K108A/R213A Mge1 and Ssc1 indicated that the disruption of contact at this site destabilized the interaction between the two proteins. We propose that the direct interaction between the loop of Ssc1 and Mge1 is not required to effect nucleotide release but plays a role in stabilization of the Mge1-Ssc1 interaction. The robust growth of the K108A/R213A MGE1 mutant suggests that the interaction between Mge1 and Ssc1 is tighter than required for functionin vivo. Molecular chaperones such as members of the 70-kDa class (Hsp70s) 1The abbreviations used are: Hsp70, 70-kDa class heat shock protein; PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.1The abbreviations used are: Hsp70, 70-kDa class heat shock protein; PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase. bind to nonnative conformations of proteins thus facilitating cellular processes such as folding of proteins and their translocation across membranes (1Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3101) Google Scholar, 2Johnson J.L. Craig E.A. Cell. 1997; 90: 201-204Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Although the C-terminal 28-kDa region of Hsp70s binds unfolded polypeptides, the highly conserved N-terminal 44-kDa domain regulates that binding through its interaction with adenine nucleotides. It is thought that Hsp70 proteins, like many GTPases, have a two-state conformation. When an ADP molecule is bound to the nucleotide-binding site, the Hsp70 exhibits stable peptide binding; when ATP is bound, binding of peptide is relatively unstable (3Schmid D. Baici A. Gehring H. Christen P. Science. 1994; 263: 971-973Crossref PubMed Scopus (420) Google Scholar, 4Theyssen H. Schuster H.P. Packschies L. Bukau B. Reinstein J. J. Mol. Biol. 1996; 263: 657-670Crossref PubMed Scopus (200) Google Scholar). The 44-kDa domain also has a low intrinsic ATPase activity (5McKay D. Wilbanks S. Flaherty K. Ha J.-H. O'Brien M. Shirvanee L. Morimoto R. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 153-178Google Scholar); therefore, ATP hydrolysis converts Hsp70 to the form having a relatively stable interaction with unfolded proteins. However, exchange of ADP for ATP results in transient interactions. Nucleotide release factors are essential components of at least some Hsp70 chaperone machines. GrpE of Escherichia coli was the first nucleotide release factor identified (6Liberek K. Marszalek J. Ang D. Georgopoulos C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Crossref PubMed Scopus (685) Google Scholar). Release of ADP from a DnaK-ADP complex can be increased up to 5000-fold by GrpE action, resulting in a reduction in the affinity of DnaK for ADP of about 200-fold (7Packschies L. Theyssen H. Buchberger A. Bukau B. Goody R. Reinstein J. Biochemistry. 1997; 36: 3417-3422Crossref PubMed Scopus (153) Google Scholar). A related protein, Mge1, an essential protein of yeast mitochondria (8Laloraya S. Gambill B.D. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6481-6485Crossref PubMed Scopus (137) Google Scholar, 9Bolliger L. Deloche O. Glick B. Georgopoulos C. Jeno P. Kronidou N. Horst M. Morishima N. Schatz G. EMBO J. 1994; 13: 1998-2006Crossref PubMed Scopus (142) Google Scholar, 10Ikeda E. Yoshida S. Mitsuzawa H. Uno I. Toh-e A. FEBS Lett. 1994; 339: 265-268Crossref PubMed Scopus (69) Google Scholar), has recently been shown to stimulate nucleotide release from the mitochondrial Hsp70, Ssc1 (11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google Scholar, 12Dekker P.J. Pfanner N. J. Mol. Biol. 1997; 270: 321-327Crossref PubMed Scopus (44) Google Scholar). Interaction of both proteins with their respective Hsp70 is resistant to high salt but disrupted by addition of ATP (11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google Scholar, 13Zylicz M. Ang D. Georgopoulos C. J. Biol. Chem. 1987; 262: 17437-17442Abstract Full Text PDF PubMed Google Scholar). GrpE binds stably to the 44-kDa N-terminal domain, although there are likely interactions with the C-terminal region of the protein as well (14Buchberger A. Schroder H. Buttner M. Valencia A. Bukau B. Nat. Struct. Biol. 1994; 1: 95-101Crossref PubMed Scopus (113) Google Scholar, 15Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (410) Google Scholar). GrpE is a homodimer that binds a single DnaK molecule. The binding is asymmetric, with one of the monomers providing the vast majority of interactive sites with the 44-kDa N terminus. The 44-kDa is composed of two large domains, each of which is composed of two subdomains. Subdomains IA and IIA lie at the base of the deep ATP-binding cleft; subdomains IB and IIB form the ATP/ADP-binding sites. There are six areas of interaction spread across one face of the 44-kDa fragment. Comparison of GrpE-related sequences suggests that the structures of GrpE and Mge1 are similar, as are their interactions with Hsp70s (16Deloche O. Georgopoulos C. J. Biol. Chem. 1996; 271: 23960-23966Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In fact, Mge1 is able to substitute for GrpE in E. coli (17DeLoche O. Kelley W. Georgopoulos C. J. Bacteriol. 1997; 179: 6066-6075Crossref PubMed Google Scholar). One of these six sites of interaction between GrpE and DnaK is a conserved loop (amino acids 28–34 and 56–62 in domain IA of DnaK and Ssc1, respectively) which was found to be important in GrpE-DnaK and Mge1-Ssc1 interactions (11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google Scholar, 14Buchberger A. Schroder H. Buttner M. Valencia A. Bukau B. Nat. Struct. Biol. 1994; 1: 95-101Crossref PubMed Scopus (113) Google Scholar, 15Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (410) Google Scholar). Designated as site IV in the published structure, this loop is not a major site in terms of overall contact area in the GrpE:DnaK co-crystal structure (15Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (410) Google Scholar) (Fig. 1). However, a mutation that resulted in an alanine instead of a glycine at position 32 (G32A) caused a destabilization of the interaction between DnaK and GrpE. The G32A mutant protein was unable to rescue either the growth defect of cells lacking wild-type DnaK or their inability to replicate phage λ (14Buchberger A. Schroder H. Buttner M. Valencia A. Bukau B. Nat. Struct. Biol. 1994; 1: 95-101Crossref PubMed Scopus (113) Google Scholar). It was proposed that this destabilization of the physical interaction prevented GrpE from facilitating nucleotide release, hence the null phenotype. The analysis of the analogous alteration in Ssc1 (G60D) presented a more confusing picture. The mutant Ssc1 stably bound Mge1, but nucleotide release was not stimulated by this interaction (11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google Scholar). The ineffectual binding of Mge1 to the G60D mutant suggested that interaction of Mge1 with this loop of region IV was important for triggering nucleotide release. To address the role of the loop region of Ssc1 in the mechanism of nucleotide release we constructed and analyzed alanine substitution mutants expected to alleviate all Mge1 interaction with Ssc1 at site IV. The results of the analysis indicate that this site of interaction between Mge1 and Ssc1 plays a role in the stabilization of the interaction between the two proteins but is not critical in the facilitation of nucleotide release. The Saccharomyces cerevisiae and E. colistrains used in this study are listed in Table I. LB medium was prepared as described (18Ausubel F. Brent R. Kingston R. Moore D. Seidman J.G. Smith J. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1997Google Scholar) and supplemented with 100 μg/ml ampicillin, 100 μg/ml kanamycin, and/or 25 μg/ml chloramphenicol where appropriate. Yeast YPD and minimal media were prepared as described (19Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar).Table IBacterial and yeast strainsStrainGenotype and phenotypeSource or Ref.E. coliPK101MG1655, F−,dnaK14 dnaJ1533Kang P.J. Craig E.A. J. Bacteriol. 1990; 172: 2055-2064Crossref PubMed Scopus (149) Google ScholarOD212AM267, dnaK332 ΔgrpE::omega-camR17DeLoche O. Kelley W. Georgopoulos C. J. Bacteriol. 1997; 179: 6066-6075Crossref PubMed Google ScholarDA259C600,thr::Tn 10Ikeda E. Yoshida S. Mitsuzawa H. Uno I. Toh-e A. FEBS Lett. 1994; 339: 265-268Crossref PubMed Scopus (69) Google ScholargrpEΔ::omega-camR kanR28DA810B178, pheA::Tn10 grpE28027Ang D. Chandrasekhar G.N. Zylicz M. Georgopoulos C. J. Bacteriol. 1986; 167: 25-29Crossref PubMed Google ScholarRLM569C600, hsdR leu-pro-lac-tonA rpsL (strR) recA22Karzai A.W. McMacken R. J. Biol. Chem. 1996; 271: 11236-11246Abstract Full Text Full Text PDF PubMed Scopus (202) Google ScholarS. cerevisiaeBJ3497pep4:HIS3 ura3–52 his3Δ20034Jones E.W. Methods Enzymol. 1991; 194: 428-453Crossref PubMed Scopus (366) Google ScholarJD100lys2 ura3–52 Δtrp1 leu2–3, 112 ssc1–1(LEU2)11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google ScholarBM37–7trp1–1 ura3–1 leu2–3,112 his3–11,15 ade2–1 can1–100 GAL2+ met2-Δ1 lys2-Δ2 SSC1ΔClaI::LEU2 mge1–2 [pRS316K-SSC1-MGE1]This study Open table in a new tab pBW401 is a low copy plasmid, pWSK29 (20Wang R.F. Kushner S.R. Gene (Amst.). 1991; 100: 195-199Crossref PubMed Scopus (1005) Google Scholar), containing the wild-type grpE gene (21Wu B. Ang D. Snavely M. Georgopoulos C. J. Bacteriol. 1994; 176: 6965-6973Crossref PubMed Google Scholar). A control plasmid, pWSK29ΔS.E, was constructed by cleaving pBW401 with SalI and EcoRI and religating to remove the grpE gene. Mutant grpE genes were constructed and cloned into pWSK29 replacing the wild-type sequences with the mutant sequences, generating pWSK29grpEK82A, pWSK29grpER183A, and pWSK29grpEK82A/R183A. pRLM232 harbors the dnaJgene under the control of the λ pL and pRpromoters, both of which are controlled by the thermosensitive λcI repressor encoded by the plasmid (22Karzai A.W. McMacken R. J. Biol. Chem. 1996; 271: 11236-11246Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). The plasmids used in the purification of Mge1, pGEX-KT-MGE1, and the GST-Ssc1 fusion, pRD56CS-SSC1, were described previously (11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google Scholar). Mutant MGE1genes were cloned into pGEX-KT-MGE1 to generate the plasmids pGEX-KT-mge1-K108A, pGEX-KT-mge1-R213A, and pGEX-KT-mge1-K108A/R213A which were used to purify mutant Mge1 proteins. Plasmid pRS314-SSC1-MGE1 was generated from pRS314-SSC1 (11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google Scholar). Mutations K108A, R213A, and K108A/R213A in MGE1 and K82A, R183A, and K82A/R183A in grpE were generated by PCR by using a standard two-step PCR procedure (23Cormack B. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 1. John Wiley & Sons, Inc., New York1994: 8.5.7-8.5.9Google Scholar). Primers used to generate the mutants are as follows: K82A, 5′-CTGGATATTGAAGCTGCCCACAAAT-3′; K108A, 5′-AGGATATTCAGAAAGCTAAGGACT-3′; R183A, 5′GCTAAATGGTGCTACGATTCGTG-3′; R213A, 5′-TCACCTTGAATGACNNNGTTATCAGACCAGCAAAAGTC-3′. The mutated fragments were cloned into appropriate plasmids and sequenced to verify the presence of the desired mutation and the absence of other mutations. To test for the ability of mutant Mge1 proteins to rescue the inviability of strains lacking Mge1, BM37-7 carrying plasmid pRS316-SSC1-MGE1 was transformed with various pRS314-SSC1-MGE1 plasmids that carry the wild-type MGE1 gene or mutant variants. Transformants were selected on minimal media lacking tryptophan; colonies were then patched onto the same medium containing 5-fluoroorotic acid (Toronto Research Chemicals, Inc.) to select for cells having lost the pRS316 plasmid containing the URA3gene and wild-type SSC1 and MGE1 genes but retaining the pRS314 plasmid containing the TRP1, wild-typeSSC1, and mutant MGE1 genes. Cells were tested on YPD plates at 30 and 37 °C. E. coli strains OD212, DA259, and DA810 were grown in L broth supplemented with appropriate antibiotics and transformed with pWSK29ΔSE and variants containing mutant grpE genes or lacking a grpE gene. Transformants were selected by plating on medium containing appropriate antibiotics. Growth of transformants was tested by plating serial dilutions of cultures onto plates containing L broth supplemented with the appropriate antibiotics; growth was observed after overnight incubation at various temperatures. Mutant and wild-type Mge1 were purified essentially as described previously (11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google Scholar). Briefly, E. coli PK101 cells harboring the expression plasmid pGEX-KT-MGE1 were grown at 30 °C and induced for expressed by the addition of 0.1 mmisopropyl-β-d-thiogalactopyranoside. 3 h after addition of isopropyl-β-d-thiogalactopyranoside, cells were harvested and disrupted with a French pressure cell, and the soluble extract was incubated with glutathione-agarose beads (Sigma). After washing, the beads were incubated with thrombin (Sigma T3010); the cleavage product was collected. The protein preparations were judged to be greater than 95% pure by Coomassie Blue staining. The protein concentrations of preparations were determined using the Bradford assay (Bio-Rad) using ovalbumin as a standard. Ssc1 was purified from yeast strain BJ3497 carrying the expression plasmid pRD56CS-SSC1 which expresses a GST-SSC1 fusion. The purification procedure was essentially the same as that used for preparation of Mge1 (11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google Scholar), except Ssc1 was further purified using DEAE-Sepharose chromatography after cleavage with thrombin. DnaJ expression was induced in RLM569 carrying the plasmid pRLM232 as described by Karzai and McMacken (22Karzai A.W. McMacken R. J. Biol. Chem. 1996; 271: 11236-11246Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). DnaJ was purified as described in Zylicz et al. (24Zylicz M. Yamamoto T. McKittrick Sell S. Georgopolous C. J. Biol. Chem. 1985; 260: 7591-7598Abstract Full Text PDF PubMed Google Scholar) using DEAE-Sephacel, followed by ammonium sulfate precipitation and hydroxylapatite chromatography. DnaK was purifed as described by Kamath-Loeb et al. (25Kamath-Loeb A. Lu C.Z. Suh W.-C. Lonetto M. Gross C. J. Biol. Chem. 1995; 270: 30051-30059Crossref PubMed Scopus (62) Google Scholar), using a DEAE-Sepharose column, followed by ATP-agarose and hydroxylapatite chromatography. 1 μl of a 1 mg/ml solution of luciferase (Sigma) dissolved in 1 m glycylglycine was added to 5.4 μl of unfolding buffer (25 mm Hepes-KOH (pH 7.5), 50 mm KCl, 5 mm MgCl2, 5 mm β-mercaptoethanol, and 6 m guanidine HCl) and incubated 1 h at room temperature. 0.078 μg of the unfolded luciferase (1 μl) was mixed with 62 μl of refolding buffer (25 mm Hepes-KOH (pH 7.5), 50 mm KCl, 5 mm MgCl2, 1 mm dithiothreitol and 1 mm ATP) which was 0.8 μm DnaK and varying amounts of DnaJ or Mge1 and incubated at room temperature. At various times 2 μl of the reactions were rapidly diluted 1:25 with dilution buffer (25 mm Hepes-KOH (pH 7.5), 50 mm KCl, 5 mm MgCl2, 1 mm dithiothreitol, and 0.1 mg/ml bovine serum albumin), injected into a luminometer (Berthold), and the activity of luciferase was measured. Mitochondria were prepared from BM37-7 containing various pRS314-SSC1-MGE1 plasmids as described previously (26Gambill B.D. Voos W. Kang P.J. Miao B. Langer T. Craig E.A. Pfanner N. J. Cell Biol. 1993; 123: 109-117Crossref PubMed Scopus (219) Google Scholar) and stored at −70 °C until use. 100 μg of mitochondria was suspended in P80 buffer (10 mm MOPS-KOH (pH 7.2), 250 mm sucrose, 80 mm KCl, 5 mm MgCl2, 3% (w/v) bovine serum albumin) and then centrifuged at 14,000 rpm for 7 min at 4 °C. Pellets were lysed on ice by incubation in lysis buffer A (250 mm sucrose, 80 mm KCl, 20 mm MOPS-KOH (pH 7.2), 0.2% Triton X-100, and 5 mm EDTA) or lysis buffer B (250 mmsucrose, 80 mm KCl, 20 mm MOPS-KOH (pH 7.2), 0.2% Triton X-100, 3 mm Mg(OAc)2 and 1 mm ATP) for 15 min. Lysates were centrifuged for 10 min at 14,000 rpm at 4 °C. Aliquots were mixed with 10 μl (bed volume) of protein A-Sepharose beads (Sigma) cross-linked with purified anti-Ssc1 antibody and incubated with the supernatants of the lysates for 1 h at 4 °C. The beads were washed with lysis buffer A or B 3 times. Beads were resuspended in 2× Laemmli's buffer (24 mmTris-HCl (pH 6.8), 10% glycerol, 0.8% SDS, 5.76 mmβ-mercaptoethanol, and 0.04% bromphenol blue). The resuspended proteins were separated by SDS-12.5% PAGE, blotted, and probed with antibodies against Mge1 and Ssc1. ECL Western blots (Amersham Pharmacia Biotech) were performed according to manufacturer's suggestions. GST-Ssc1 fusion protein was immobilized on glutathione-agarose beads as described previously (11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google Scholar). After extensive washing with buffer D (25 mm Hepes-KOH (pH 7.4), 50 mm KCl, 10% glycerol, 1 mm EDTA), an equal volume of 0.1 μm Mge1 was added to the beads and incubated at 4 °C for 1 h. The beads were then sequentially washed with buffer D, buffer D containing 1 m KCl, buffer D and eluted with buffer D containing 10 mm Mg(OAc)2 and 10 mmATP. Equivalent samples were collected at various stages, separated by SDS-PAGE, and probed with antibodies as described above. Complex formation and single turnover assay were performed essentially as described previously (11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google Scholar) with several modifications. 25 μg of wild-type or mutant Ssc1 was incubated with 100 μCi of [α-32P]ATP (DuPont, 3000 Ci/mmol) in buffer E (25 mm Hepes-KOH (pH 7.5), 100 mm KCl, and 11 mm Mg(OAc)2) containing 25 μm ATP at 30 °C for 15 min. The reaction was chilled on ice and immediately loaded onto a NICK column (Amersham Pharmacia Biotech) pre-equilibrated with buffer E at 4 °C. 70-μl fractions were collected. The first peak of radioactivity that corresponded to the Ssc1-ATP complex was pooled, adjusted to 10% glycerol, aliquoted, and stored at −70 °C. For a single turnover assay, a 10-μl aliquot of the Ssc1-ATP complex was thawed and immediately mixed with an equal volume of buffer E containing other components such as Mge1, as indicated, and incubated at 30 °C. At the indicated times the reaction was stopped and the percent conversion to ADP determined (11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google Scholar). The loop encoded by amino acids 56–62 of Ssc1 was previously implicated in the nucleotide release activity of Mge1 (11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google Scholar). Mutant proteins with certain single amino acid substitutions such as G60D were able to bind Mge1, but nucleotide release was not observed. We reasoned that if the G60D mutation disrupted a critical interaction with Mge1, it might be possible to partially overcome this defect by alterations in Mge1 that could promote nucleotide release in the Mge1-G60D Ssc1 complex. Therefore we attempted to isolate suppressor mutations inMGE1 which rescued the lethality of the G60D mutant. Two strategies were used. MGE1 was randomly mutagenized over its entire length using error-prone PCR. In addition, codon Arg-213 was targeted for mutagenesis because the side chain of this highly conserved encoded arginine is predicted to interact with Gly-60 (Fig.1) (15Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (410) Google Scholar). Oligonucleotides that could encode each possible amino acid at codon 213 were used to create a library of MGE1 genes containing alterations at this residue. No suppressors were identified by either method (data not shown). Since no mutations in MGE1 were identified that could overcome the defect of the SSC1 G60D mutation, we decided to test directly the importance of residue Arg-213. Six single amino acid substitutions were tested by transforming a plasmid carrying the mutantMGE1 genes into a strain having a deletion ofMGE1 on the chromosome and a wild-type copy ofMGE1 on a URA3-containing plasmid. The ability of the mutant MGE1 to rescue growth of a mge1deletion mutant was monitored by streaking on 5-fluoroorotic acid-containing media which inhibits growth of cells expressing theURA3 gene product. Therefore only cells carrying mutant genes which were able to support growth in the absence of expression of wild-type MGE1 were able to form colonies in this test. Of the six mutations tested, five (R213A, R213L, R213C, R213D, and R213H) allowed wild-type growth at 30 °C (Fig.2 and data not shown). Only R213P was nonfunctional. The effect of the proline substitution is not surprising because of the propensity of proline to substantially disrupt structure. Inspection of the GrpE:DnaK structure suggests that the only other interaction between this loop and Mge1 occurs between Lys-108 of Mge1 and Glu-56 of Ssc1. These residues are identical in GrpE and DnaK. Therefore we constructed mutations at the Lys-108 codon ofMGE1 by site-directed mutagenesis and tested their ability to function. Alanine was chosen as a substitution because it would be expected to disrupt the normal interaction with the side chain of Glu-56 in Ssc1; glutamic acid was selected since it would change a positive to a negative charge at this position, and would be predicted to juxtapose two amino acids having the same charge. However, strains carrying either mutant gene grew as well as wild-type cells at a variety of temperatures, utilizing both fermentable and nonfermentable carbon sources (Fig. 2 and data not shown). Since each alanine substitution alone caused no detectable phenotype, we constructed the double mutant K108A/R213A MGE1 to test the effect of alanines at both positions. Based on the GrpE:DnaK structure these mutations would be expected to obviate all interactions between Mge1 and the loop of Ssc1 since the side chains of the Mge1 amino acids are the interactive sites (Fig. 1). This double mutant was viable, growing as well as wild-type at 30 °C and only slightly compromised for growth at 37 °C on both glucose- and glycerol-based medium (Fig. 2 and data not shown). These results suggest that the physical interaction between Mge1 and the loop of Ssc1 is not essential for Mge1 to function as a nucleotide exchange factor. The interpretation of the results presented above assumes that the Mge1-Ssc1 interaction is the same as the GrpE-DnaK interaction. Although the amino acid sequences of the two pairs of proteins are related and the amino acids relevant to the interaction discussed here are identical, it is not known whether the two structures are completely analogous. Therefore, we decided to change amino acids at the positions in GrpE (Lys-82 and Arg-183), which are analogous to those tested in Mge1, to alanines and test the effect of these substitutions on GrpE function. Three systems have been established for analysis of mutations in grpE, an essential gene as follows: the rescue of the temperature-sensitive growth of 1) thegrpE mutation, grpE280 (17DeLoche O. Kelley W. Georgopoulos C. J. Bacteriol. 1997; 179: 6066-6075Crossref PubMed Google Scholar, 27Ang D. Chandrasekhar G.N. Zylicz M. Georgopoulos C. J. Bacteriol. 1986; 167: 25-29Crossref PubMed Google Scholar); 2) a strain carrying both a deletion of the grpE gene and thednaK332 compensatory allele which allows growth at lower temperatures in the absence of GrpE (17DeLoche O. Kelley W. Georgopoulos C. J. Bacteriol. 1997; 179: 6066-6075Crossref PubMed Google Scholar); and 3) a strain carrying an extragenic suppressor of the grpE deletion mutation (28Ang D. Georgopoulos C. J. Bacteriol. 1989; 17: 2748-2755Crossref Google Scholar). We tested the single and the double mutants by transforming low copy plasmids carrying each mutant gene into appropriate strains and testing for growth at 30, 37, 42, and 43 °C. For comparison, each test strain was also transformed with a plasmid containing wild-typegrpE. As can be seen in Fig. 3all the mutants restored growth of the test strains to that observed in the presence of wild-type grpE. Therefore, Lys-82 and Arg-183 of GrpE, like the analogous residues in Mge1, are not essential for in vivo function. Hence, since the nucleotide release activity is thought to be the essential function of GrpE, these results suggest that these residues are not essential for the nucleotide release activity of these factors. In addition, these results also support the idea that the Mge1-Ssc1 interaction and the GrpE-DnaK interaction are the same. Since the double mutant K108A/R213A allowed nearly wild-type growth, we tested its ability to stimulate the release of ATP from Ssc1 as the nucleotide release activity is thought to be the essential function of GrpE/Mge1. To assess release activity we used a single turnover ATPase assay, monitoring the hydrolysis of radiolabeled ATP prebound to Ssc1(11). This assay is based on the idea that release of nucleotide from Ssc1 facilitated by Mge1 will cause a decrease in hydrolysis of the radiolabeled ATP. Since an excess of unlabeled ATP is included in the reaction, released radiolabeled nucleotide will only rarely be rebound to Ssc1. The radiolabeled adenine nucleotide content of the isolated nucleotide-Ssc1 complex was about 80% ATP and 20% ADP (Fig.4 A). About 45% of the ATP bound to Ssc1 was hydrolyzed within 10 min at 30 °C. As expected, addition of nonradioactive ATP to the reaction had little effect on the hydrolysis of the prebound ATP indicating that the nucleotide remains bound throughout the time course of the reaction. However, as previously reported (11Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (81) Google Scholar), addition of wild-type Mge1 reduced the hydrolysis of radiolabeled ATP when unlabeled nucleotide was added to prevent rebinding of released radiolabeled nucleotide (Fig.4 B). For example, the presence of Mge1 at a concentration of 8 μm reduced ATP hydrolysis by about 70% at 10 min incubation. A significant lowering of hydrolysis was observed upon addition of Mge1 to concentrations as low as 2 μm; at this concentration of Mge1 a 28% reduction was observed. On the other hand, no effect on ATP hydrolysis was observed upon addition of 8 μm K108A/R213A Mge1 (Fig. 4 C). However, addition of higher amounts of mutant Mge1 resulted in a reduction in hydrolysis. For example, a 24% reduction in hydrolysis occurred upon ad
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