Abstract
Nucleotide binding to the 70 kDa heat-shock cognate protein (Hsc70) from mung bean seeds and pig brain was investigated, as well as the clathrin uncoating activity of Hsc70 in the presence of these nucleotides. The two enzymes were found to behave identically. ATP bound to two different forms of Hsc70, with dissociation constants of 1.1 +/- 0.1 microM and 1.4 +/- 0.7 mM respectively at 25 degrees C. This corresponds to delta G0' = -34 and -16 kJ/mol respectively. From the temperature-dependence of the dissociation constant of the high-affinity site, delta H0' was calculated to -36 +/- 2 kJ/mol. This gives delta S0' = 6.7 J/mol per K. Adenosine 5'-[gamma-thio]triphosphate, ADP, adenosine 5'-[beta, gamma-imino]triphosphate and adenosine 5'-[beta, gamma-methylene]triphosphate showed dissociation constants of 2.3, 11, 31 and 284 microM respectively. The order of affinities corresponded to the order of effectiveness in uncoating of pig brain coated vesicles. The implications of these findings for the mechanism of Hsc70 action are discussed.
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- Blond-Elguindi S., Fourie A. M., Sambrook J. F., Gething M. J. Peptide-dependent stimulation of the ATPase activity of the molecular chaperone BiP is the result of conversion of oligomers to active monomers. J Biol Chem. 1993 Jun 15;268(17):12730–12735. [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Braell W. A., Schlossman D. M., Schmid S. L., Rothman J. E. Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase. J Cell Biol. 1984 Aug;99(2):734–741. doi: 10.1083/jcb.99.2.734. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Buxbaum E., Woodman P. G. Selective action of uncoating ATPase towards clathrin-coated vesicles from brain. J Cell Sci. 1995 Mar;108(Pt 3):1295–1306. doi: 10.1242/jcs.108.3.1295. [DOI] [PubMed] [Google Scholar]
- Buxbaum E., Woodman P. G. The speed of partial reactions of the uncoating ATPase Hsc70 depends on the source of coated vesicles. J Cell Sci. 1996 Mar;109(Pt 3):705–711. doi: 10.1242/jcs.109.3.705. [DOI] [PubMed] [Google Scholar]
- Chappell T. G., Konforti B. B., Schmid S. L., Rothman J. E. The ATPase core of a clathrin uncoating protein. J Biol Chem. 1987 Jan 15;262(2):746–751. [PubMed] [Google Scholar]
- Flaherty K. M., DeLuca-Flaherty C., McKay D. B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature. 1990 Aug 16;346(6285):623–628. doi: 10.1038/346623a0. [DOI] [PubMed] [Google Scholar]
- Freiden P. J., Gaut J. R., Hendershot L. M. Interconversion of three differentially modified and assembled forms of BiP. EMBO J. 1992 Jan;11(1):63–70. doi: 10.1002/j.1460-2075.1992.tb05028.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gao B., Emoto Y., Greene L., Eisenberg E. Nucleotide binding properties of bovine brain uncoating ATPase. J Biol Chem. 1993 Apr 25;268(12):8507–8513. [PubMed] [Google Scholar]
- Gao B., Greene L., Eisenberg E. Characterization of nucleotide-free uncoating ATPase and its binding to ATP, ADP, and ATP analogues. Biochemistry. 1994 Mar 1;33(8):2048–2054. doi: 10.1021/bi00174a010. [DOI] [PubMed] [Google Scholar]
- Georgopoulos C. The emergence of the chaperone machines. Trends Biochem Sci. 1992 Aug;17(8):295–299. doi: 10.1016/0968-0004(92)90439-g. [DOI] [PubMed] [Google Scholar]
- Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. doi: 10.1016/0003-2697(89)90602-7. [DOI] [PubMed] [Google Scholar]
- Ha J. H., McKay D. B. ATPase kinetics of recombinant bovine 70 kDa heat shock cognate protein and its amino-terminal ATPase domain. Biochemistry. 1994 Dec 6;33(48):14625–14635. doi: 10.1021/bi00252a031. [DOI] [PubMed] [Google Scholar]
- Hartl F. U., Hlodan R., Langer T. Molecular chaperones in protein folding: the art of avoiding sticky situations. Trends Biochem Sci. 1994 Jan;19(1):20–25. doi: 10.1016/0968-0004(94)90169-4. [DOI] [PubMed] [Google Scholar]
- Heuser J., Steer C. J. Trimeric binding of the 70-kD uncoating ATPase to the vertices of clathrin triskelia: a candidate intermediate in the vesicle uncoating reaction. J Cell Biol. 1989 Oct;109(4 Pt 1):1457–1466. doi: 10.1083/jcb.109.4.1457. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
- Palleros D. R., Reid K. L., Shi L., Welch W. J., Fink A. L. ATP-induced protein-Hsp70 complex dissociation requires K+ but not ATP hydrolysis. Nature. 1993 Oct 14;365(6447):664–666. doi: 10.1038/365664a0. [DOI] [PubMed] [Google Scholar]
- Palleros D. R., Welch W. J., Fink A. L. Interaction of hsp70 with unfolded proteins: effects of temperature and nucleotides on the kinetics of binding. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5719–5723. doi: 10.1073/pnas.88.13.5719. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Perkins S. J. Protein volumes and hydration effects. The calculations of partial specific volumes, neutron scattering matchpoints and 280-nm absorption coefficients for proteins and glycoproteins from amino acid sequences. Eur J Biochem. 1986 May 15;157(1):169–180. doi: 10.1111/j.1432-1033.1986.tb09653.x. [DOI] [PubMed] [Google Scholar]
- Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
- Rothman J. E., Schmid S. L. Enzymatic recycling of clathrin from coated vesicles. Cell. 1986 Jul 4;46(1):5–9. doi: 10.1016/0092-8674(86)90852-4. [DOI] [PubMed] [Google Scholar]
- Sadis S., Hightower L. E. Unfolded proteins stimulate molecular chaperone Hsc70 ATPase by accelerating ADP/ATP exchange. Biochemistry. 1992 Oct 6;31(39):9406–9412. doi: 10.1021/bi00154a012. [DOI] [PubMed] [Google Scholar]
- Schlossman D. M., Schmid S. L., Braell W. A., Rothman J. E. An enzyme that removes clathrin coats: purification of an uncoating ATPase. J Cell Biol. 1984 Aug;99(2):723–733. doi: 10.1083/jcb.99.2.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schmid S. L., Braell W. A., Rothman J. E. ATP catalyzes the sequestration of clathrin during enzymatic uncoating. J Biol Chem. 1985 Aug 25;260(18):10057–10062. [PubMed] [Google Scholar]
- Schmid S. L., Rothman J. E. Enzymatic dissociation of clathrin cages in a two-stage process. J Biol Chem. 1985 Aug 25;260(18):10044–10049. [PubMed] [Google Scholar]
- Schu P. V., Takegawa K., Fry M. J., Stack J. H., Waterfield M. D., Emr S. D. Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science. 1993 Apr 2;260(5104):88–91. doi: 10.1126/science.8385367. [DOI] [PubMed] [Google Scholar]
- Straume M., Johnson M. L. Monte Carlo method for determining complete confidence probability distributions of estimated model parameters. Methods Enzymol. 1992;210:117–129. doi: 10.1016/0076-6879(92)10009-3. [DOI] [PubMed] [Google Scholar]
- Toledo H., Carlino A., Vidal V., Redfield B., Nettleton M. Y., Kochan J. P., Brot N., Weissbach H. Dissociation of glucose-regulated protein Grp78 and Grp78-IgE Fc complexes by ATP. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2505–2508. doi: 10.1073/pnas.90.6.2505. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ungewickell E. The 70-kd mammalian heat shock proteins are structurally and functionally related to the uncoating protein that releases clathrin triskelia from coated vesicles. EMBO J. 1985 Dec 16;4(13A):3385–3391. doi: 10.1002/j.1460-2075.1985.tb04094.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang C., Lee M. R. High-level expression of soluble rat hsc70 in Escherichia coli: purification and characterization of the cloned enzyme. Biochem J. 1993 Aug 15;294(Pt 1):69–77. doi: 10.1042/bj2940069. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang C., Lin B. L. The disappearance of an hsc70 species in mung bean seed during germination: purification and characterization of the protein. Plant Mol Biol. 1993 Jan;21(2):317–329. doi: 10.1007/BF00019947. [DOI] [PubMed] [Google Scholar]