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. 1994 Mar 29;91(7):2743–2747. doi: 10.1073/pnas.91.7.2743

Primary structure and function of a second essential member of the heterooligomeric TCP1 chaperonin complex of yeast, TCP1 beta.

D Miklos 1, S Caplan 1, D Mertens 1, G Hynes 1, Z Pitluk 1, Y Kashi 1, K Harrison-Lavoie 1, S Stevenson 1, C Brown 1, B Barrell 1, et al.
PMCID: PMC43446  PMID: 7908441

Abstract

A role for heterooligomeric TCP1 complex as a chaperonin in the eukaryotic cytosol has recently been suggested both by structural similarities with other chaperonins and by in vitro experiments showing it to mediate ATP-dependent folding of actin, tubulin, and luciferase. Here we present the primary structure of a second subunit of the complex and present genetic and functional analyses. The TCP1 beta amino acid sequence, predicted from the cloned gene, bears 35% identity to TCP1, termed here TCP1 alpha, containing the same highly conserved residues found in the collective sequence of chaperonins. The predicted product was identified as the fastest-migrating species of the TCP1 complex purified from soluble extracts of yeast. The TCP1 beta gene, like TCP1 alpha, is essential. Strains containing lethal disruptions of either gene could not be rescued by additional copies of the other. Spores bearing disruption of either gene germinated as single, large-budded cells. Similarly, large-budded cells were observed following shift to 37 degrees C of strains carrying temperature-sensitive mutations in either TCP1 alpha or TCP1 beta. The arrested cells contained replicated DNA present in single nuclear masses, associated with abnormal tubulin staining patterns, supporting the assertion that mitotic spindle formation and function are impaired. We conclude that TCP1 beta supplies an essential function that partially overlaps with that of TCP1 alpha in acting as a molecular chaperone in tubulin and spindle biogenesis.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barraclough R., Ellis R. J. Protein synthesis in chloroplasts. IX. Assembly of newly-synthesized large subunits into ribulose bisphosphate carboxylase in isolated intact pea chloroplasts. Biochim Biophys Acta. 1980 Jun 27;608(1):19–31. doi: 10.1016/0005-2787(80)90129-x. [DOI] [PubMed] [Google Scholar]
  2. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  3. Bond J. F., Fridovich-Keil J. L., Pillus L., Mulligan R. C., Solomon F. A chicken-yeast chimeric beta-tubulin protein is incorporated into mouse microtubules in vivo. Cell. 1986 Feb 14;44(3):461–468. doi: 10.1016/0092-8674(86)90467-8. [DOI] [PubMed] [Google Scholar]
  4. Braig K., Simon M., Furuya F., Hainfeld J. F., Horwich A. L. A polypeptide bound by the chaperonin groEL is localized within a central cavity. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3978–3982. doi: 10.1073/pnas.90.9.3978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cheng M. Y., Hartl F. U., Martin J., Pollock R. A., Kalousek F., Neupert W., Hallberg E. M., Hallberg R. L., Horwich A. L. Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature. 1989 Feb 16;337(6208):620–625. doi: 10.1038/337620a0. [DOI] [PubMed] [Google Scholar]
  6. Ellis R. J. Molecular chaperones: the plant connection. Science. 1990 Nov 16;250(4983):954–959. doi: 10.1126/science.250.4983.954. [DOI] [PubMed] [Google Scholar]
  7. Ellis R. J., van der Vies S. M. Molecular chaperones. Annu Rev Biochem. 1991;60:321–347. doi: 10.1146/annurev.bi.60.070191.001541. [DOI] [PubMed] [Google Scholar]
  8. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frydman J., Nimmesgern E., Erdjument-Bromage H., Wall J. S., Tempst P., Hartl F. U. Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits. EMBO J. 1992 Dec;11(13):4767–4778. doi: 10.1002/j.1460-2075.1992.tb05582.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gao Y., Thomas J. O., Chow R. L., Lee G. H., Cowan N. J. A cytoplasmic chaperonin that catalyzes beta-actin folding. Cell. 1992 Jun 12;69(6):1043–1050. doi: 10.1016/0092-8674(92)90622-j. [DOI] [PubMed] [Google Scholar]
  11. Georgopoulos C. P., Hendrix R. W., Casjens S. R., Kaiser A. D. Host participation in bacteriophage lambda head assembly. J Mol Biol. 1973 May 5;76(1):45–60. doi: 10.1016/0022-2836(73)90080-6. [DOI] [PubMed] [Google Scholar]
  12. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  13. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  14. Gupta R. S. Sequence and structural homology between a mouse T-complex protein TCP-1 and the 'chaperonin' family of bacterial (GroEL, 60-65 kDa heat shock antigen) and eukaryotic proteins. Biochem Int. 1990;20(4):833–841. [PubMed] [Google Scholar]
  15. Hemmingsen S. M., Woolford C., van der Vies S. M., Tilly K., Dennis D. T., Georgopoulos C. P., Hendrix R. W., Ellis R. J. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature. 1988 May 26;333(6171):330–334. doi: 10.1038/333330a0. [DOI] [PubMed] [Google Scholar]
  16. Hendrick J. P., Hartl F. U. Molecular chaperone functions of heat-shock proteins. Annu Rev Biochem. 1993;62:349–384. doi: 10.1146/annurev.bi.62.070193.002025. [DOI] [PubMed] [Google Scholar]
  17. Hendrix R. W. Purification and properties of groE, a host protein involved in bacteriophage assembly. J Mol Biol. 1979 Apr 15;129(3):375–392. doi: 10.1016/0022-2836(79)90502-3. [DOI] [PubMed] [Google Scholar]
  18. Hohn T., Hohn B., Engel A., Wurtz M., Smith P. R. Isolation and characterization of the host protein groE involved in bacteriophage lambda assembly. J Mol Biol. 1979 Apr 15;129(3):359–373. doi: 10.1016/0022-2836(79)90501-1. [DOI] [PubMed] [Google Scholar]
  19. Horwich A. L., Willison K. R. Protein folding in the cell: functions of two families of molecular chaperone, hsp 60 and TF55-TCP1. Philos Trans R Soc Lond B Biol Sci. 1993 Mar 29;339(1289):313–326. doi: 10.1098/rstb.1993.0030. [DOI] [PubMed] [Google Scholar]
  20. Huffaker T. C., Thomas J. H., Botstein D. Diverse effects of beta-tubulin mutations on microtubule formation and function. J Cell Biol. 1988 Jun;106(6):1997–2010. doi: 10.1083/jcb.106.6.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kilmartin J. V., Adams A. E. Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J Cell Biol. 1984 Mar;98(3):922–933. doi: 10.1083/jcb.98.3.922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kubota H., Hynes G., Carne A., Ashworth A., Willison K. Identification of six Tcp-1-related genes encoding divergent subunits of the TCP-1-containing chaperonin. Curr Biol. 1994 Feb 1;4(2):89–99. doi: 10.1016/s0960-9822(94)00024-2. [DOI] [PubMed] [Google Scholar]
  23. Langer T., Pfeifer G., Martin J., Baumeister W., Hartl F. U. Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity. EMBO J. 1992 Dec;11(13):4757–4765. doi: 10.1002/j.1460-2075.1992.tb05581.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lewis V. A., Hynes G. M., Zheng D., Saibil H., Willison K. T-complex polypeptide-1 is a subunit of a heteromeric particle in the eukaryotic cytosol. Nature. 1992 Jul 16;358(6383):249–252. doi: 10.1038/358249a0. [DOI] [PubMed] [Google Scholar]
  25. McMullin T. W., Hallberg R. L. A highly evolutionarily conserved mitochondrial protein is structurally related to the protein encoded by the Escherichia coli groEL gene. Mol Cell Biol. 1988 Jan;8(1):371–380. doi: 10.1128/mcb.8.1.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Melki R., Vainberg I. E., Chow R. L., Cowan N. J. Chaperonin-mediated folding of vertebrate actin-related protein and gamma-tubulin. J Cell Biol. 1993 Sep;122(6):1301–1310. doi: 10.1083/jcb.122.6.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Novick P., Botstein D. Phenotypic analysis of temperature-sensitive yeast actin mutants. Cell. 1985 Feb;40(2):405–416. doi: 10.1016/0092-8674(85)90154-0. [DOI] [PubMed] [Google Scholar]
  28. Olson M. V., Dutchik J. E., Graham M. Y., Brodeur G. M., Helms C., Frank M., MacCollin M., Scheinman R., Frank T. Random-clone strategy for genomic restriction mapping in yeast. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7826–7830. doi: 10.1073/pnas.83.20.7826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Reading D. S., Hallberg R. L., Myers A. M. Characterization of the yeast HSP60 gene coding for a mitochondrial assembly factor. Nature. 1989 Feb 16;337(6208):655–659. doi: 10.1038/337655a0. [DOI] [PubMed] [Google Scholar]
  30. Rose M. D., Fink G. R. KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell. 1987 Mar 27;48(6):1047–1060. doi: 10.1016/0092-8674(87)90712-4. [DOI] [PubMed] [Google Scholar]
  31. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  32. Sefton B. M., Campbell M. A. The role of tyrosine protein phosphorylation in lymphocyte activation. Annu Rev Cell Biol. 1991;7:257–274. doi: 10.1146/annurev.cb.07.110191.001353. [DOI] [PubMed] [Google Scholar]
  33. Segel G. B., Boal T. R., Cardillo T. S., Murant F. G., Lichtman M. A., Sherman F. Isolation of a gene encoding a chaperonin-like protein by complementation of yeast amino acid transport mutants with human cDNA. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6060–6064. doi: 10.1073/pnas.89.13.6060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sternberg N. Properties of a mutant of Escherichia coli defective in bacteriophage lambda head formation (groE). I. Initial characterization. J Mol Biol. 1973 May 5;76(1):1–23. doi: 10.1016/0022-2836(73)90078-8. [DOI] [PubMed] [Google Scholar]
  36. Sternlicht H., Farr G. W., Sternlicht M. L., Driscoll J. K., Willison K., Yaffe M. B. The t-complex polypeptide 1 complex is a chaperonin for tubulin and actin in vivo. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9422–9426. doi: 10.1073/pnas.90.20.9422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Trent J. D., Nimmesgern E., Wall J. S., Hartl F. U., Horwich A. L. A molecular chaperone from a thermophilic archaebacterium is related to the eukaryotic protein t-complex polypeptide-1. Nature. 1991 Dec 12;354(6353):490–493. doi: 10.1038/354490a0. [DOI] [PubMed] [Google Scholar]
  38. Ursic D., Culbertson M. R. Is yeast TCP1 a chaperonin? Nature. 1992 Apr 2;356(6368):392–392. doi: 10.1038/356392a0. [DOI] [PubMed] [Google Scholar]
  39. Ursic D., Culbertson M. R. The yeast homolog to mouse Tcp-1 affects microtubule-mediated processes. Mol Cell Biol. 1991 May;11(5):2629–2640. doi: 10.1128/mcb.11.5.2629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vida T. A., Graham T. R., Emr S. D. In vitro reconstitution of intercompartmental protein transport to the yeast vacuole. J Cell Biol. 1990 Dec;111(6 Pt 2):2871–2884. doi: 10.1083/jcb.111.6.2871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. West A. H., Clark D. J., Martin J., Neupert W., Hartl F. U., Horwich A. L. Two related genes encoding extremely hydrophobic proteins suppress a lethal mutation in the yeast mitochondrial processing enhancing protein. J Biol Chem. 1992 Dec 5;267(34):24625–24633. [PubMed] [Google Scholar]
  42. Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]
  43. Yaffe M. B., Farr G. W., Miklos D., Horwich A. L., Sternlicht M. L., Sternlicht H. TCP1 complex is a molecular chaperone in tubulin biogenesis. Nature. 1992 Jul 16;358(6383):245–248. doi: 10.1038/358245a0. [DOI] [PubMed] [Google Scholar]

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