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• W2015918623 abstract "The multisite-specific endonuclease Endo.SceI of yeast mitochondria is unique among endonucleases because its 50-kDa subunit forms a stable dimer with the mitochondrial 70-kDa heat shock protein (mtHSP70), which otherwise fulfills a chaperone function by binding transiently to unfolded proteins. Here we show that the mtHSP70 subunit confers broader sequence specificity, greater stability, and higher activity on the 50-kDa subunit. The 50-kDa subunit alone displayed weaker activity and highly sequence-specific endonuclease activity. The 50-kDa protein exists as a heterodimer with mtHSP70 in vivo, allowing Endo.SceI to cleave specifically at multiple sites on mitochondrial DNA. Endo.SceI may have evolved from a highly specific endonuclease that gained broader sequence specificity after becoming a stable partner of mtHSP70. The multisite-specific endonuclease Endo.SceI of yeast mitochondria is unique among endonucleases because its 50-kDa subunit forms a stable dimer with the mitochondrial 70-kDa heat shock protein (mtHSP70), which otherwise fulfills a chaperone function by binding transiently to unfolded proteins. Here we show that the mtHSP70 subunit confers broader sequence specificity, greater stability, and higher activity on the 50-kDa subunit. The 50-kDa subunit alone displayed weaker activity and highly sequence-specific endonuclease activity. The 50-kDa protein exists as a heterodimer with mtHSP70 in vivo, allowing Endo.SceI to cleave specifically at multiple sites on mitochondrial DNA. Endo.SceI may have evolved from a highly specific endonuclease that gained broader sequence specificity after becoming a stable partner of mtHSP70. mitochondrial 70-kDa heat shock protein 70-kDa heat shock protein reduced carboxymethylated α-lactalbumin glutathione S-transferase base pair kilobase pair Endo.SceI is a yeast mitochondrial endonuclease having multisequence specificity (1Watabe H. Shibata T. Ando T. J. Biochem. (Tokyo). 1981; 90: 1623-1632Crossref PubMed Scopus (17) Google Scholar, 2Shibata T. Watabe H. Kaneko T. Iino T. Ando T. J. Biol. Chem. 1984; 259: 10499-10506Abstract Full Text PDF PubMed Google Scholar, 3Morishima N. Nakagawa K. Yamamoto E. Shibata T. J. Biol. Chem. 1990; 265: 15189-15197Abstract Full Text PDF PubMed Google Scholar). Endo.SceI was purified as a heterodimer comprised of 50- and 75-kDa subunits (4Nakagawa K. Hashikawa J. Makino O. Ando T. Shibata T. Eur. J. Biochem. 1988; 171: 23-29Crossref PubMed Scopus (9) Google Scholar), the latter of which was later identified as the mitochondrial 70-kDa heat shock protein (mtHSP70,1 also referred as Ssc1p) (3Morishima N. Nakagawa K. Yamamoto E. Shibata T. J. Biol. Chem. 1990; 265: 15189-15197Abstract Full Text PDF PubMed Google Scholar). The 50-kDa subunit shares the consensus amino acid sequence LAGLIDADG (5Nakagawa K. Morishima N. Shibata T. J. Biol. Chem. 1991; 266: 1977-1984Abstract Full Text PDF PubMed Google Scholar, 6Belfort M. Roberts R.J. Nucleic Acids Res. 1997; 25: 3379-3388Crossref PubMed Scopus (392) Google Scholar) for yeast sequence-specific endonucleases involved in genetic recombination (e.g. HO endonuclease and mitochondrial ω endonuclease), suggesting that this subunit is responsible for the core activity of the endonuclease. The recognition sequences of recombinational endonucleases are as long as 17–26 bp (7Kostriken R. Strathern J.N. Klar A.J.S. Hicks J.B. Heffron F. Cell. 1983; 35: 167-174Abstract Full Text PDF PubMed Scopus (192) Google Scholar, 8Colleaux L. D'Auriol L. Betermier M. Cottarel G. Jacquier A. Galibert F. Dujon B. Cell. 1986; 44: 521-533Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 9Delahodde A. Goguel V. Becam A.M. Creusot F. Perea J. Banroques J. Jacq C. Cell. 1989; 56: 431-441Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 10Kawasaki K. Takahashi M. Natori M. Shibata T. J. Biol. Chem. 1991; 266: 5342-5347Abstract Full Text PDF PubMed Google Scholar) and are unisequence-specific, which means that they cleave at only one unique site on the whole genome. The Endo.SceI endonuclease is unique among these endonucleases in that it tolerates degeneracy at several positions within the cleavage sequence (2Shibata T. Watabe H. Kaneko T. Iino T. Ando T. J. Biol. Chem. 1984; 259: 10499-10506Abstract Full Text PDF PubMed Google Scholar). Because of its broad sequence specificity, Endo.SceI cleaves randomly at over 30 sites on yeast mitochondrial DNA both in vitro and in vivo (Ref. 11Shibata T. Nakagawa K. Morishima N. Adv. Biophys. 1995; 31: 77-91Crossref PubMed Scopus (7) Google Scholar and data shown below). The function of Endo.SceI is important during the process of mitochondrial fusion when haploid cells are mated to form zygotic cells. During this process, Endo.SceI cleaves mitochondrial DNA to induce genetic recombination among the heterogeneous mitochondrial DNAs inherited from the parents (12Nakagawa K.-I. Morishima N. Shibata T. EMBO J. 1992; 11: 2707-2715Crossref PubMed Scopus (43) Google Scholar).The 50-kDa protein is not the sole binding partner of mtHSP70. mtHSP70 is a member of the HSP70 family and has broad affinity for proteins in the unfolded state. Molecular genetics and biochemical analysis have revealed that mtHSP70 transiently binds to nuclear-encoded mitochondrial proteins which become unfolded during their transfer from the cytosol across the mitochondrial membrane. mtHSP70 is essential for the import of these proteins into mitochondria and their subsequent folding (13Scherer P.E. Krieg U.C. Hwang S.T. Vestweber D. Schatz G. EMBO J. 1990; 9: 4315-4322Crossref PubMed Scopus (163) Google Scholar, 14Kang P.J. Ostermann J. Shilling J. Neupert W. Craig E.A. Pfanner N. Nature. 1990; 348: 137-143Crossref PubMed Scopus (539) Google Scholar). More than 10 homologs of HSP70 have been detected or cloned from the genome sequence of the yeast Saccharomyces cerevisiae (15Mukai H. Kuno T. Tanaka H. Hirata D. Miyakawa T. Tanaka C. Gene (Amst.). 1993; 132: 57-66Crossref PubMed Scopus (101) Google Scholar, 16Shirayama M. Kawakami K. Matsui Y. Tanaka K. Toh-e A. Mol. Gen. Genet. 1993; 240: 323-332Crossref PubMed Scopus (42) Google Scholar, 17Craig E.A. Baxter B.K. Becker J. Halladay J. Ziegehoffer T. Morimoto R.I. Tissières A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 31-52Google Scholar, 18Craven R.A. Egerton M. Stirling C.J. EMBO J. 1996; 15: 2640-2650Crossref PubMed Scopus (141) Google Scholar, 19Schilke B. Forster J. Davis J. James P. Walter W. Laloraya S. Johnson J. Miao B. Craig E. J. Cell Biol. 1996; 134: 603-613Crossref PubMed Scopus (70) Google Scholar). The HSP70 family functions in a diverse set of processes, including regulation of the heat shock response, quality control of proteins and protein folding, as well as protein translocation across organellar membranes (20Morimoto R.I. Tissières A. Georgopoulos C. Morimoto R.I. Tissières A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994: 1-30Google Scholar, 21Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3085) Google Scholar).The 50-kDa protein is unique among the binding partners of HSP70 in that mtHSP70 does not chaperone the 50-kDa protein as a transient partner but becomes stably incorporated as part of the heterodimer complex. The diverse functions of HSP70 described above are accomplished through transient binding of HSP70 to proteins in an unfolded state. HSP70 prevents the unfolded proteins from aggregating and misfolding, until the proteins are either properly folded, transferred into organelles, or degraded by proteases (21Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3085) Google Scholar). The chaperone function of mtHSP70 and the sequence motifs found in the 50-kDa subunit led us to speculate that mtHSP70 might somehow modulate the structure and activity of the 50-kDa subunit. It has been difficult, however, to examine if mtHSP70 has a regulatory role for the endonuclease activity because the heterodimer is so stable under nondenaturing conditions that the 50-kDa protein cannot be functionally analyzed in the absence of mtHSP70 (4Nakagawa K. Hashikawa J. Makino O. Ando T. Shibata T. Eur. J. Biochem. 1988; 171: 23-29Crossref PubMed Scopus (9) Google Scholar).Standard overexpression systems have not been applied to the 50-kDa protein because it is encoded by a mitochondrial gene whose translation requires mitochondrial specific codon usage. To examine the role of mtHSP70 in the heterodimer, we established overexpression and purification systems for the 50-kDa subunit. To our surprise, the purified 50-kDa protein by itself showed endonuclease activity that was not multisequence-specific but unisequence-specific. The Endo.SceI heterodimer was easily reconstructed by incubating the 50-kDa protein with mtHSP70 in the presence of ADP. The 50-kDa protein did not appear to bind to the substrate binding domain of mtHSP70 but to the ATPase domain. By comparing the enzyme activities of the 50-kDa protein with the reconstructed heterodimer, we found that mtHSP70 converts the unisequence-specific 50-kDa endonuclease into a multisequence-specific enzyme. Thus, multisequence specificity, the characteristic feature of Endo.SceI, is dependent on the stable binding of mtHSP70.RESULTSOverexpression of the 50-kDa Subunit in E. coliThe 50-kDa subunit is encoded by a mitochondrial gene whose coding sequence contains 37 mitochondrial specific codons (5Nakagawa K. Morishima N. Shibata T. J. Biol. Chem. 1991; 266: 1977-1984Abstract Full Text PDF PubMed Google Scholar). The 50-kDa subunit gene was modified so that the whole amino acid sequence could be directed by universal codons and expressed in widely used overexpression systems. Briefly, 13 cycles of site-directed mutagenesis (22Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4884) Google Scholar) were performed by using mutagenic oligonucleotide primers. Up to 16 primers were used at a time to reduce the number of mutagenesis cycles, unless the primer sequences overlapped with one another. For easier purification of the 50-kDa protein, its N terminus was tagged with a hexahistidine sequence. We overexpressed the hexahistidine-tagged 50-kDa protein in E. coli and purified it by affinity chromatography and ion exchange chromatography (Fig.1 A). Since DnaK, a bacterial HSP70, is present in E. coli cells, the 50-kDa protein sample was examined for contamination with DnaK. Western blot analysis with an anti-DnaK monoclonal antibody showed that bacterial HSP70 could not be detected in the 50-kDa protein sample (Fig. 1 A). For reconstruction of Endo.SceI in vitro, mtHSP70 was purified from the isolated mitochondria of yeast cells which overexpress mtHSP70 (Fig. 1 A) as reported previously (25Bolliger L. Deloche O. Glick B.S. Georgopolous C. Jeno P. Kronidou N. Horst M. Morishima N. Schatz G. EMBO J. 1994; 13: 1998-2006Crossref PubMed Scopus (141) Google Scholar). We used a yeast strain that lacked the functional gene for the 50-kDa protein to avoid possible contamination of the mtHSP70 sample with the 50-kDa protein (5Nakagawa K. Morishima N. Shibata T. J. Biol. Chem. 1991; 266: 1977-1984Abstract Full Text PDF PubMed Google Scholar, 12Nakagawa K.-I. Morishima N. Shibata T. EMBO J. 1992; 11: 2707-2715Crossref PubMed Scopus (43) Google Scholar).The quaternary structure of the purified 50-kDa protein was analyzed by gel filtration chromatography. The purified 50-kDa protein was eluted as a single symmetrical peak between the elution positions of bovine serum albumin (67 kDa) and ovalbumin(43 kDa), suggesting a globular and monomeric structure (Fig. 1 B).The Purified 50-kDa Protein Is a Unisequence-specific EndonucleaseIn general, the function of HSP70 is accomplished through its transient binding to unfolded or denatured proteins (13Scherer P.E. Krieg U.C. Hwang S.T. Vestweber D. Schatz G. EMBO J. 1990; 9: 4315-4322Crossref PubMed Scopus (163) Google Scholar, 14Kang P.J. Ostermann J. Shilling J. Neupert W. Craig E.A. Pfanner N. Nature. 1990; 348: 137-143Crossref PubMed Scopus (539) Google Scholar, 28Beckmann R.P. Mizzen L.E. Welch W.J. Science. 1990; 248: 850-854Crossref PubMed Scopus (1040) Google Scholar, 29Skowyra D. Georgopoulos C. Zylicz M. Cell. 1990; 62: 939-944Abstract Full Text PDF PubMed Scopus (327) Google Scholar). According to this rationale, mtHSP70 binding to the 50 kDa would occur because the chaperone function of mtHSP70 recognizes unfolded structures in the 50-kDa subunit. However, the gel filtration profile of the 50-kDa protein suggests that it is a globular protein of 50 kDa with a properly folded structure. To analyze further the functional and structural nature of the 50-kDa protein, we measured the circular dichroism spectrum and endonuclease activity of the subunit.The monomeric form of the 50-kDa protein showed a typical spectrum for α-helix containing proteins with two minima at 208 and 222 nm (Fig.1 C). The value of the ellipticity suggested that the percentage of α-helix content in the 50-kDa protein was around 38% with no indication of an unusually high percentage of random structures (30Greenfield N. Fasman G.D. Biochemistry. 1969; 8: 4108-4116Crossref PubMed Scopus (3300) Google Scholar). This result, together with the gel filtration pattern, suggests that the 50-kDa protein has a native structure and intrinsic endonuclease activity in the absence of the mtHSP70 subunit.Actually we found that the 50-kDa protein alone can cleave a specific sequence within the mitochondrial oli2 gene cloned into pUC119 (Fig. 1 D). This specific site in the oli2 gene has been shown to be cleaved by Endo.SceI in vivo, and cleavage at this site induces efficient gene conversion between heterologous mitochondrial DNAs inherited from parental cells during mating (12Nakagawa K.-I. Morishima N. Shibata T. EMBO J. 1992; 11: 2707-2715Crossref PubMed Scopus (43) Google Scholar). The sizes of the cleaved products (3.4 and 1.4 kb) suggest that the cutting site of the 50-kDa protein was identical to that of the Endo.SceI dimer. The 50-kDa protein did not digest the oli2 mutant sequence (12Nakagawa K.-I. Morishima N. Shibata T. EMBO J. 1992; 11: 2707-2715Crossref PubMed Scopus (43) Google Scholar) containing an altered Endo.SceI cutting site (data not shown). These results, as well as the physical properties of the 50-kDa protein, indicate that the 50-kDa protein possesses a native structure and acts as a functional endonuclease. However, the 50-kDa protein did not exhibit multisequence specificity which is a characteristic feature of the Endo.SceI heterodimer. The 50-kDa protein could not cleave plasmid DNAs, phage DNAs, or yeast mitochondrial DNA except for the oli2 sequence, whereas all of these DNAs have been used to detect Endo.SceI endonuclease activity (see below). For instance, the purified 50-kDa protein did not cleave a cutting site in pBR322 DNA which has been used in the standard assay for Endo.SceI (2Shibata T. Watabe H. Kaneko T. Iino T. Ando T. J. Biol. Chem. 1984; 259: 10499-10506Abstract Full Text PDF PubMed Google Scholar, 4Nakagawa K. Hashikawa J. Makino O. Ando T. Shibata T. Eur. J. Biochem. 1988; 171: 23-29Crossref PubMed Scopus (9) Google Scholar). These data suggested that mtHSP70 possesses a novel function distinct from its normal role in protein folding.Reconstruction of the Endo.SceI HeterodimerTo investigate the role of mtHSP70 in the Endo.SceI heterodimer, we tried to reconstruct the heterodimer from each subunit in vitro. HSP70 can be found in three states depending on the nature of the bound nucleotide as follows: ATP-bound HSP70, ADP-bound HSP70, and nucleotide-free HSP70. In general, ADP-bound HSP70 makes a more stable complex with unfolded proteins than ATP-bound HSP70. The dissociation constants of ADP-bound HSP70 with substrates are between 5- and 85-fold lower than those of ATP-bound HSP70, although the ADP-bound form has slower binding rates for substrates (for a review, see Ref. 31Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2400) Google Scholar). As an initial trial, the active 50-kDa protein was incubated with ADP-bound mtHSP70 without prior denaturing treatment of the endonuclease. We chose 25 °C as the temperature for the complex formation reaction because the 50-kDa protein loses little activity in terms of its ability to cleave within the oli2 sequence when incubated at this temperature even after several hours (data not shown). After the 50-kDa subunit was incubated with a stoichiometric amount of ADP-mtHSP70, the protein mixture was loaded onto a gel filtration column to analyze complex formation. The 50-kDa subunit was detected in fractions from the column by Western blot analysis and the endonuclease assay. The elution profile suggested that the Endo.SceI heterodimer was efficiently formed in vitro from the 50-kDa protein and mtHSP70, as the position of the peak fraction (fraction 68) corresponded to a molecular size of around 125 kDa (Fig.2). It should be noted that dimer formation did not require denaturing treatment of the 50-kDa protein. The endonuclease assay using the oli2 substrate revealed that the Endo.SceI activity could only be observed in the fraction containing the heterodimer. No complexes of the 50-kDa subunit appeared at the elution volume of the heterodimer when the 50-kDa subunit was incubated alone (data not shown). The nucleotide dependence of heterodimer formation, as well as the nature of the binding between the 50-kDa protein and mtHSP70, will be described below.Figure 2Heterodimer formation of the 50-kDa protein and mtHSP70 in vitro. A mixture of the 50-kDa protein and mtHSP70 was incubated at 25 °C for 90 min and then loaded onto a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech). The elution positions of marker proteins are indicated by arrowheads (aldolase, 158-kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa). The endonuclease activity of the eluted proteins was assayed by using pMITO DNA (upper panel). Eluted fractions were also analyzed by immunoblot analysis with the anti-6xHis monoclonal antibody (lower panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of mtHSP70 Binding on the Endonuclease Activity of the 50-kDa SubunitSubstratesAfter establishing the conditions for complex formation, we quantitatively analyzed the effect of mtHSP70 binding on the activity of the 50-kDa protein. Two DNAs were chosen as substrates in the nuclease assay. The first one contained the mitochondrial oli2 sequence, which is the natural substrate efficiently cleaved by Endo.SceI in vivo (12Nakagawa K.-I. Morishima N. Shibata T. EMBO J. 1992; 11: 2707-2715Crossref PubMed Scopus (43) Google Scholar) and the 50-kDa protein in vitro (Fig. 1 D). The other substrate contained the Endo.SceI-cleavable sequence of pBR322 (2Shibata T. Watabe H. Kaneko T. Iino T. Ando T. J. Biol. Chem. 1984; 259: 10499-10506Abstract Full Text PDF PubMed Google Scholar). Since the cleavage sequence of Endo.SceI is not unique but multiple, we determined the consensus sequence for Endo.SceI cleavage by comparing the cleavable sequences found in phage and plasmid DNAs (2Shibata T. Watabe H. Kaneko T. Iino T. Ando T. J. Biol. Chem. 1984; 259: 10499-10506Abstract Full Text PDF PubMed Google Scholar) (Fig.3 A). Unlike bacterial restriction enzymes, Endo.SceI recognizes and binds to at least 26-bp regions of DNA (10Kawasaki K. Takahashi M. Natori M. Shibata T. J. Biol. Chem. 1991; 266: 5342-5347Abstract Full Text PDF PubMed Google Scholar). The cleavable sequences of oli2 and pBR322 used in the present experiment contained four and six mismatches to the consensus sequence, respectively (Fig. 3 A).Figure 3Binding of mtHSP70 to the 50-kDa protein confers greater stability and broader sequence specificity. A, the cleavage sites for the Endo.SceI dimer.Top, the consensus sequence for the Endo.SceI cleavage as previously determined (2Shibata T. Watabe H. Kaneko T. Iino T. Ando T. J. Biol. Chem. 1984; 259: 10499-10506Abstract Full Text PDF PubMed Google Scholar, 10Kawasaki K. Takahashi M. Natori M. Shibata T. J. Biol. Chem. 1991; 266: 5342-5347Abstract Full Text PDF PubMed Google Scholar). R represents either G or A, Y either C or T, and N corresponds to a degenerate position. Middle, the cleavage site in the mitochondrial oli2 region (12Nakagawa K.-I. Morishima N. Shibata T. EMBO J. 1992; 11: 2707-2715Crossref PubMed Scopus (43) Google Scholar). Bottom, the cleavage site found in pBR322 (2Shibata T. Watabe H. Kaneko T. Iino T. Ando T. J. Biol. Chem. 1984; 259: 10499-10506Abstract Full Text PDF PubMed Google Scholar). B, stability of the endonuclease activity. The 50-kDa protein (1 μg/ml) with or without ADP-mtHSP70 (1.5 μg/ml) was preincubated at 37 °C for each indicated time in the assay mixture without the substrate, and then the reaction was started by the addition of the oli2 plasmid. Without preincubation, the substrate was partially cleaved (up to about 80%) by the endonuclease. C, enhancement of the endonuclease activity. The 50-kDa protein with or without ADP-mtHSP70 was incubated with the oli2 substrate at 37 °C for 1 min. The amount of the 50-kDa protein used is shown in the figure. For comparison, the activity of the endogenous enzyme (Endo.SceI) is also plotted in the figure. D, the endonuclease activity of the reconstructed Endo.SceI heterodimer. The pBR322 substrate (pBR1) was cleaved by the reconstituted dimer (lanes 1–5). The amounts of 50-kDa protein as heterodimer in the digestion were 0 (lane 1), 0.125 (lane 2), 0.25 (lane 3), 0.50 (lane 4), and 1.0 μg/ml (lane 5). Major cleavage products are indicated by arrowheads on the left side. Cleavage at minor sites within the vector backbone (2Shibata T. Watabe H. Kaneko T. Iino T. Ando T. J. Biol. Chem. 1984; 259: 10499-10506Abstract Full Text PDF PubMed Google Scholar) was also evident after longer incubation. pBR1 was not cleaved by the 50-kDa protein alone (lanes 6–10). The amounts of the monomeric form of the 50-kDa protein were 0 (lane 6), 2 (lane 7), 4 (lane 8), 8 (lane 9), and 16 μg/ml (lane 10). Lane M denotes a Hin dIII digest of phage λ DNA. E, specific interaction of the 50-kDa protein and mtHSP70 is required for cleavage of the pBR322 substrate. The pBR322 substrate (pBR1) was incubated with various combinations of proteins. The 50-kDa protein used for the digestion was 1.0 μg/ml. Either mtHSP70 or E. coli DnaK that had been loaded with ADP was incubated with the 50-kDa protein at a 1:1 molar ratio prior to digestion. F, modulation of sequence specificity of the 50-kDa protein by mtHSP70. Mitochondrial DNA was completely digested with Bam HI and Pst I and then treated with the 50-kDa protein, either with or without mtHSP70. The amount of the monomeric 50-kDa protein was 5.3 μg/ml, whereas 1.0 μg/ml of the 50-kDa protein was used for digestion in the presence of mtHSP70. The DNA fragments generated by the cleavage at the oli2 sequence are indicated by arrowheads.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Thermostability and Specific ActivityWe have observed that Endo.SceI is a heat-sensitive enzyme that loses its activity at 37 °C within 2 h (1Watabe H. Shibata T. Ando T. J. Biochem. (Tokyo). 1981; 90: 1623-1632Crossref PubMed Scopus (17) Google Scholar). 2H. Mizumura, T. Shibata, and N. Morishima, unpublished data. Thus, we expected that the 50-kDa protein would be even more heat-labile. Since one of the biological functions of HSP70 is to protect unfolded proteins from aggregation and irreversible inactivation (for a review, see Ref. 21Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3085) Google Scholar), we examined the thermostability of the 50-kDa protein in the presence or absence of mtHSP70. After preincubation of the 50-kDa protein at 37 °C the endonuclease reaction was done for 1 min to compensate for the possible inactivation of the 50-kDa protein during the reaction (note that the standard assay for Endo.SceI uses 30–60 min for digestion). Fig. 3 B shows that the endonuclease activity of both the 50-kDa monomer and the reconstructed Endo.SceI decreased during the preincubation at 37 °C. However, the 50-kDa protein displayed reduced thermostability in the absence of the other subunit. After 15 min of preincubation, the residual activity of the 50-kDa monomer was less than 40% of the original activity, and it further decreased below 20% after 30 min preincubation. Under the same conditions, the reconstructed dimer was more resistant to inactivation, as its endonuclease activity remained stable with over 80% of the original activity remaining after 30 min of preincubation. These results indicate that the 50-kDa protein is heat-labile and that the mtHSP70 subunit confers thermoresistance on the 50-kDa protein. In a separate experiment, addition of mtHSP70 to the 50-kDa protein inactivated at 37 °C did not restore the endonuclease activity, suggesting that the loss of activity under these conditions is due to irreversible inactivation.We next examined the specific activity of the 50-kDa protein with or without mtHSP70, as we observed that the endonuclease activity of the reconstituted dimer was always higher than that of the 50-kDa monomer. The assay was again done for 1 min because this short assay time contributed little to the time difference in heat inactivation between the monomer and the Endo.SceI dimer as shown by the above results. Fig.3 C shows an approximately 3–4-fold enhancement of endonuclease activity for the reconstructed dimer up to a concentration of 1 μg/ml. The specific activity of the reconstituted dimer is comparable to that of the endogenous Endo.SceI heterodimer within the similar range of protein concentrations (Fig. 3 C). These results indicate that the binding of mtHSP70 to the 50-kDa protein confers higher specific activity as well as greater thermostability.Sequence SpecificityWith the oli2 DNA, the endonuclease activity of the monomeric 50-kDa subunit was approximately one-third that of the heterodimer (Fig. 3 C). This suggests that we might be able to detect cleavages of other substrate DNAs by the 50-kDa subunit at similar efficiency unless the multiple sequence specificity is different from that of the Endo.SceI dimer. Preliminary experiments showed that the 50-kDa protein could not cleave pBR322, although Endo.SceI can specifically cleave it. A simple explanation for the result is that the apparent change in sequence specificity might be the result of mtHSP70 enhancement of the overall activity of the 50-kDa protein. Another possibility is that the 50-kDa protein has very narrow sequence specificity compared with the reconstituted mtHSP70/50-kDa heterodimer. To examine these possibilities, we quantitatively compared the endonuclease activity of the reconstructed dimer with that of the 50-kDa protein using the pBR322 substrate.In agreement with previous observations of Endo.SceI endonuclease specificity, Fig. 3 D shows that the reconstructed dimer cleaved the pBR322 substrate as well as the oli2 sequence (2Shibata T. Watabe H. Kaneko T. Iino T. Ando T. J. Biol. Chem. 1984; 259: 10499-10506Abstract Full Text PDF PubMed Google Scholar, 12Nakagawa K.-I. Morishima N. Shibata T. EMBO J. 1992; 11: 2707-2715Crossref PubMed Scopus (43) Google Scholar). However, the pBR322 substrate was not cleaved by the 50-kDa subunit at all. Cleavage of the pBR322 substrate was not observed even at concentrations of up to 16 μg/ml of the 50-kDa protein, whereas the cleavage of the same substrate was evident with only 0.125 μg/ml of the 50-kDa protein included in the reconstituted dimer (Fig.3 D). Neither the 50-kDa protein alone nor mtHSP70 showed endonuclease activity on the pBR322 substrate (Fig. 3 E), indicating that the 50-kDa protein gained multiple sequence specificity on association with mtHSP70. The interaction between the 50-kDa protein and mtHSP70 appears to be specific because the 50-kDa protein preincubated with ADP-loaded DnaK at a 1:1 molar ratio did not show cleavage activity on the pBR322 substrate (Fig.3 E).To compare the sequence specificity of the reconstructed dimer with that of the 50-kDa monomer in a more comprehensive manner, we used yeast mitochondrial DNA as a substrate for the endonuclease assay. We prepared Bam HI/Pst I double restriction digests of mitochondrial DNA, which was then treated with either the 50-kDa protein or Endo.SceI. The mitochondrial DNA used has a unique Pst I site within the 50-kDa protein gene, which is located at about 1.2 kb downstream of the Endo.SceI cleavage site in the oli2 gene (5Nakagawa K. Morishima N. Shibata T. J. Biol. Chem. 1991; 266: 1977-1984Abstract Full Text PDF PubMed Google Scholar, 12Nakagawa K.-I. Morishima N. Shibata T. EMBO J. 1992; 11: 2707-2715Crossref PubMed Scopus (43) Google Scholar). Fig. 3 F shows that the 50-kDa protein monomer (5.3 μg/ml) generated fragments of 1.2 and 8.4 kb from the mitochondrial DNA, indicating specific cleavage at the oli2 sequence. No additional fragment appeared even at concentrations up to 10.6 μg/ml of the 50-kDa protein (data not shown). This result favors the idea that the oli2 gene is the unique cleavage site for the 50-kDa protein within mitochondrial DNA. In contrast to the apparent unisequence specificity of the 50-kDa protein, the Endo.SceI heterodimer at a concentration equivalent to 1" @default.
• W2015918623 created "2016-06-24" @default.
• W2015918623 creator A5002353805 @default.
• W2015918623 creator A5070636104 @default.
• W2015918623 creator A5072543767 @default.
• W2015918623 date "1999-09-01" @default.
• W2015918623 modified "2023-09-28" @default.
• W2015918623 title "Stable Association of 70-kDa Heat Shock Protein Induces Latent Multisite Specificity of a Unisite-specific Endonuclease in Yeast Mitochondria" @default.
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