Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Jan 1;132(1):153–165. doi: 10.1083/jcb.132.1.153

A nontetrameric species is the major soluble form of keratin in Xenopus oocytes and rabbit reticulocyte lysates

PMCID: PMC2120706  PMID: 8567720

Abstract

Inside the interphase cell, approximately 5% of the total intermediate filament protein exists in a soluble form. Past studies using velocity gradient sedimentation (VGS) indicate that soluble intermediate filament protein exists as an approximately 7 S tetrameric species. While studying intermediate filament assembly dynamics in the Xenopus oocyte, we used both VGS and size-exclusion chromatography (SEC) to analyze the soluble form of keratin. Previous studies (Coulombe, P. A., and E. Fuchs. 1990. J. Cell Biol. 111:153) report that tetrameric keratins migrate on SEC with an apparent molecular weight of approximately 150,000; the major soluble form of keratin in the oocyte, in contrast, migrates with an apparent molecular weight of approximately 750,000. During oocyte maturation, the keratin system disassembles into a soluble form (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787) and the amount of the 750-kD keratin complex increases dramatically. Immunoprecipitation analysis of soluble keratin from matured oocytes revealed the presence of type I and type II keratins, but no other stoichiometrically associated polypeptides, suggesting that the 750-kD keratin complex is composed solely of keratin. To further study the formation of the 750-kD keratin complex, we used rabbit reticulocyte lysates (RRL). The 750-kD keratin complex was formed in RRLs contranslating type I and type II Xenopus keratins, but not when lysates translated type I or type II keratin RNAs alone. The 750-kD keratin complex could be formed posttranslationally in an ATP-independent manner when type I and type II keratin translation reactions were mixed. Under conditions of prolonged incubation, such as occur during VGS analysis, the 750-kD keratin complex disassembled into a 7 S (by VGS), 150-kD (by SEC) form. In urea denaturation studies, the 7 S/150-kD form could be further disassembled into an 80-kD species that consists of cofractionating dimeric and monomeric keratin. Based on these results, the 750-kD species appears to be a supratetrameric complex of keratins and is the major, soluble form of keratin in both prophase and M-phase oocytes, and RRL reactions.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Aebi U., Fowler W. E., Rew P., Sun T. T. The fibrillar substructure of keratin filaments unraveled. J Cell Biol. 1983 Oct;97(4):1131–1143. doi: 10.1083/jcb.97.4.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blikstad I., Lazarides E. Vimentin filaments are assembled from a soluble precursor in avian erythroid cells. J Cell Biol. 1983 Jun;96(6):1803–1808. doi: 10.1083/jcb.96.6.1803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Caplan M. J., Palade G. E., Jamieson J. D. Newly synthesized Na,K-ATPase alpha-subunit has no cytosolic intermediate in MDCK cells. J Biol Chem. 1986 Feb 25;261(6):2860–2865. [PubMed] [Google Scholar]
  4. Cary R. B., Klymkowsky M. W. Differential organization of desmin and vimentin in muscle is due to differences in their head domains. J Cell Biol. 1994 Jul;126(2):445–456. doi: 10.1083/jcb.126.2.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chu D. T., Klymkowsky M. W. The appearance of acetylated alpha-tubulin during early development and cellular differentiation in Xenopus. Dev Biol. 1989 Nov;136(1):104–117. doi: 10.1016/0012-1606(89)90134-6. [DOI] [PubMed] [Google Scholar]
  6. Coulombe P. A., Fuchs E. Elucidating the early stages of keratin filament assembly. J Cell Biol. 1990 Jul;111(1):153–169. doi: 10.1083/jcb.111.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dent J. A., Cary R. B., Bachant J. B., Domingo A., Klymkowsky M. W. Host cell factors controlling vimentin organization in the Xenopus oocyte. J Cell Biol. 1992 Nov;119(4):855–866. doi: 10.1083/jcb.119.4.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eichner R., Kahn M. Differential extraction of keratin subunits and filaments from normal human epidermis. J Cell Biol. 1990 Apr;110(4):1149–1168. doi: 10.1083/jcb.110.4.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eichner R., Sun T. T., Aebi U. The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments. J Cell Biol. 1986 May;102(5):1767–1777. doi: 10.1083/jcb.102.5.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Franke W. W., Schiller D. L., Hatzfeld M., Winter S. Protein complexes of intermediate-sized filaments: melting of cytokeratin complexes in urea reveals different polypeptide separation characteristics. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7113–7117. doi: 10.1073/pnas.80.23.7113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Franke W. W., Winter S., Schmid E., Söllner P., Hämmerling G., Achtstätter T. Monoclonal cytokeratin antibody recognizing a heterotypic complex: immunological probing of conformational states of cytoskeletal proteins in filaments and in solution. Exp Cell Res. 1987 Nov;173(1):17–37. doi: 10.1016/0014-4827(87)90328-4. [DOI] [PubMed] [Google Scholar]
  14. Franz J. K., Franke W. W. Cloning of cDNA and amino acid sequence of a cytokeratin expressed in oocytes of Xenopus laevis. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6475–6479. doi: 10.1073/pnas.83.17.6475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Franz J. K., Gall L., Williams M. A., Picheral B., Franke W. W. Intermediate-size filaments in a germ cell: Expression of cytokeratins in oocytes and eggs of the frog Xenopus. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6254–6258. doi: 10.1073/pnas.80.20.6254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fuchs E., Weber K. Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem. 1994;63:345–382. doi: 10.1146/annurev.bi.63.070194.002021. [DOI] [PubMed] [Google Scholar]
  17. Gall L., Karsenti E. Soluble cytokeratins in Xenopus laevis oocytes and eggs. Biol Cell. 1987;61(1-2):33–38. doi: 10.1111/j.1768-322x.1987.tb00566.x. [DOI] [PubMed] [Google Scholar]
  18. Geisler N., Schünemann J., Weber K. Chemical cross-linking indicates a staggered and antiparallel protofilament of desmin intermediate filaments and characterizes one higher-level complex between protofilaments. Eur J Biochem. 1992 Jun 15;206(3):841–852. doi: 10.1111/j.1432-1033.1992.tb16992.x. [DOI] [PubMed] [Google Scholar]
  19. Gurdon J. B., Wickens M. P. The use of Xenopus oocytes for the expression of cloned genes. Methods Enzymol. 1983;101:370–386. doi: 10.1016/0076-6879(83)01028-9. [DOI] [PubMed] [Google Scholar]
  20. Hatzfeld M., Franke W. W. Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides. J Cell Biol. 1985 Nov;101(5 Pt 1):1826–1841. doi: 10.1083/jcb.101.5.1826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hatzfeld M., Weber K. The coiled coil of in vitro assembled keratin filaments is a heterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression. J Cell Biol. 1990 Apr;110(4):1199–1210. doi: 10.1083/jcb.110.4.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Heins S., Aebi U. Making heads and tails of intermediate filament assembly, dynamics and networks. Curr Opin Cell Biol. 1994 Feb;6(1):25–33. doi: 10.1016/0955-0674(94)90112-0. [DOI] [PubMed] [Google Scholar]
  23. Herrmann H., Eckelt A., Brettel M., Grund C., Franke W. W. Temperature-sensitive intermediate filament assembly. Alternative structures of Xenopus laevis vimentin in vitro and in vivo. J Mol Biol. 1993 Nov 5;234(1):99–113. doi: 10.1006/jmbi.1993.1566. [DOI] [PubMed] [Google Scholar]
  24. Hisanaga S., Hirokawa N. Molecular architecture of the neurofilament. II. Reassembly process of neurofilament L protein in vitro. J Mol Biol. 1990 Feb 20;211(4):871–882. doi: 10.1016/0022-2836(90)90080-6. [DOI] [PubMed] [Google Scholar]
  25. Hisanaga S., Ikai A., Hirokawa N. Molecular architecture of the neurofilament. I. Subunit arrangement of neurofilament L protein in the intermediate-sized filament. J Mol Biol. 1990 Feb 20;211(4):857–869. doi: 10.1016/0022-2836(90)90079-2. [DOI] [PubMed] [Google Scholar]
  26. Ip W., Hartzer M. K., Pang Y. Y., Robson R. M. Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments. J Mol Biol. 1985 Jun 5;183(3):365–375. doi: 10.1016/0022-2836(85)90007-5. [DOI] [PubMed] [Google Scholar]
  27. Isaacs W. B., Cook R. K., Van Atta J. C., Redmond C. M., Fulton A. B. Assembly of vimentin in cultured cells varies with cell type. J Biol Chem. 1989 Oct 25;264(30):17953–17960. [PubMed] [Google Scholar]
  28. Jonas E., Sargent T. D., Dawid I. B. Epidermal keratin gene expressed in embryos of Xenopus laevis. Proc Natl Acad Sci U S A. 1985 Aug;82(16):5413–5417. doi: 10.1073/pnas.82.16.5413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Klymkowsky M. W. Intermediate filament organization, reorganization, and function in the clawed frog Xenopus. Curr Top Dev Biol. 1995;31:455–486. doi: 10.1016/s0070-2153(08)60236-7. [DOI] [PubMed] [Google Scholar]
  30. Klymkowsky M. W., Maynell L. A., Nislow C. Cytokeratin phosphorylation, cytokeratin filament severing and the solubilization of the maternal mRNA Vg1. J Cell Biol. 1991 Aug;114(4):787–797. doi: 10.1083/jcb.114.4.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Klymkowsky M. W., Maynell L. A., Polson A. G. Polar asymmetry in the organization of the cortical cytokeratin system of Xenopus laevis oocytes and embryos. Development. 1987 Jul;100(3):543–557. doi: 10.1242/dev.100.3.543. [DOI] [PubMed] [Google Scholar]
  32. Krieg P. A., Melton D. A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 1984 Sep 25;12(18):7057–7070. doi: 10.1093/nar/12.18.7057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Liao J., Lowthert L. A., Ghori N., Omary M. B. The 70-kDa heat shock proteins associate with glandular intermediate filaments in an ATP-dependent manner. J Biol Chem. 1995 Jan 13;270(2):915–922. doi: 10.1074/jbc.270.2.915. [DOI] [PubMed] [Google Scholar]
  34. Lorimer G. H., Todd M. J., Viitanen P. V. Chaperonins and protein folding: unity and disunity of mechanisms. Philos Trans R Soc Lond B Biol Sci. 1993 Mar 29;339(1289):297–304. doi: 10.1098/rstb.1993.0028. [DOI] [PubMed] [Google Scholar]
  35. Miller R. K., Khuon S., Goldman R. D. Dynamics of keratin assembly: exogenous type I keratin rapidly associates with type II keratin in vivo. J Cell Biol. 1993 Jul;122(1):123–135. doi: 10.1083/jcb.122.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nishizawa K., Yano T., Shibata M., Ando S., Saga S., Takahashi T., Inagaki M. Specific localization of phosphointermediate filament protein in the constricted area of dividing cells. J Biol Chem. 1991 Feb 15;266(5):3074–3079. [PubMed] [Google Scholar]
  37. Okabe S., Miyasaka H., Hirokawa N. Dynamics of the neuronal intermediate filaments. J Cell Biol. 1993 Apr;121(2):375–386. doi: 10.1083/jcb.121.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pang Y. Y., Schermer A., Yu J., Sun T. T. Suprabasal change and subsequent formation of disulfide-stabilized homo- and hetero-dimers of keratins during esophageal epithelial differentiation. J Cell Sci. 1993 Mar;104(Pt 3):727–740. doi: 10.1242/jcs.104.3.727. [DOI] [PubMed] [Google Scholar]
  39. Sahyoun N., Stenbuck P., LeVine H., 3rd, Bronson D., Moncharmont B., Henderson C., Cuatrecasas P. Formation and identification of cytoskeletal components from liver cytosolic precursors. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7341–7345. doi: 10.1073/pnas.79.23.7341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Skalli O., Goldman R. D. Recent insights into the assembly, dynamics, and function of intermediate filament networks. Cell Motil Cytoskeleton. 1991;19(2):67–79. doi: 10.1002/cm.970190202. [DOI] [PubMed] [Google Scholar]
  41. Soellner P., Quinlan R. A., Franke W. W. Identification of a distinct soluble subunit of an intermediate filament protein: tetrameric vimentin from living cells. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7929–7933. doi: 10.1073/pnas.82.23.7929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Steinert P. M. Analysis of the mechanism of assembly of mouse keratin 1/keratin 10 intermediate filaments in vitro suggests that intermediate filaments are built from multiple oligomeric units rather than a unique tetrameric building block. J Struct Biol. 1991 Oct;107(2):175–188. doi: 10.1016/1047-8477(91)90020-w. [DOI] [PubMed] [Google Scholar]
  43. Steinert P. M., Liem R. K. Intermediate filament dynamics. Cell. 1990 Feb 23;60(4):521–523. doi: 10.1016/0092-8674(90)90651-t. [DOI] [PubMed] [Google Scholar]
  44. Steinert P. M. The two-chain coiled-coil molecule of native epidermal keratin intermediate filaments is a type I-type II heterodimer. J Biol Chem. 1990 May 25;265(15):8766–8774. [PubMed] [Google Scholar]
  45. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  46. Tian G., Vainberg I. E., Tap W. D., Lewis S. A., Cowan N. J. Specificity in chaperonin-mediated protein folding. Nature. 1995 May 18;375(6528):250–253. doi: 10.1038/375250a0. [DOI] [PubMed] [Google Scholar]
  47. Wallace R. A., Hollinger T. G. Turnover of endogenous, microinjected, and sequestered protein in Xenopus oocytes. Exp Cell Res. 1979 Mar 15;119(2):277–287. doi: 10.1016/0014-4827(79)90355-0. [DOI] [PubMed] [Google Scholar]
  48. 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]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES