Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Aug 1;109(2):675–683. doi: 10.1083/jcb.109.2.675

The sea urchin multicatalytic protease: purification, biochemical analysis, subcellular distribution, and relationship to snRNPs

PMCID: PMC2115700  PMID: 2527240

Abstract

We have purified and extensively characterized a 19-S particle from sea urchin eggs. This particle is the sea urchin homologue of the "prosome", a particle originally identified in duck erythroblasts. We now show that these sea urchin prosomes contain multiple proteolytic activities. As shown for analogous particles from other cells, these particles hydrolyze synthetic substrates containing neutral hydrophobic or basic amino acids at the carboxy terminus of the synthetic peptides. They contain 16-20 small proteins ranging in molecular weight from 20,000 to 32,000. Peptide mapping shows that most of the polypeptides are unique, however, three exist in two isoelectric forms. We have investigated the possible function of the sea urchin multicatalytic proteases (MCPs) by determining their subcellular distribution, their relationship to egg snRNPs, and their possible role in translational repression. There are almost as many MCPs (2 x 10(8] as ribosomes (6.6 x 10(8] or mRNPs (1.8 x 10(7] per egg. This suggests that like ribosomes, the MCPs are stored in the egg for use during later development. We find that a substantial proportion of egg MCPs move into nuclei by the late blastula stage. Using a specific antibody against one of the sea urchin MCP proteins and antibodies against U1- U6, La, and Ro RNPs, we show that the sea urchin particle is distinct from these RNPs, although the anti-U1-U6 RNP antibody cross-reacts with a single MCP protein. In addition, the sea urchin MCP appears to be associated with a large structure in the cytoplasm of unfertilized eggs and is released under the same conditions that activate egg mRNPs in vitro.

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. Akhayat O., Grossi de Sa F., Infante A. A. Sea urchin prosome: characterization and changes during development. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1595–1599. doi: 10.1073/pnas.84.6.1595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arrigo A. P., Darlix J. L., Khandjian E. W., Simon M., Spahr P. F. Characterization of the prosome from Drosophila and its similarity to the cytoplasmic structures formed by the low molecular weight heat-shock proteins. EMBO J. 1985 Feb;4(2):399–406. doi: 10.1002/j.1460-2075.1985.tb03642.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arrigo A. P., Simon M., Darlix J. L., Spahr P. F. A 20S particle ubiquitous from yeast to human. J Mol Evol. 1987;25(2):141–150. doi: 10.1007/BF02101756. [DOI] [PubMed] [Google Scholar]
  4. Arrigo A. P., Tanaka K., Goldberg A. L., Welch W. J. Identity of the 19S 'prosome' particle with the large multifunctional protease complex of mammalian cells (the proteasome). Nature. 1988 Jan 14;331(6152):192–194. doi: 10.1038/331192a0. [DOI] [PubMed] [Google Scholar]
  5. Bachmann M., Mayet W. J., Schröder H. C., Pfeifer K., Meyer zum Büschenfelde K. H., Müller W. E. Association of La and Ro antigens with intracellular structures in HEp-2 carcinoma cells. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7770–7774. doi: 10.1073/pnas.83.20.7770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Birnstiel M. L., Busslinger M., Strub K. Transcription termination and 3' processing: the end is in site! Cell. 1985 Jun;41(2):349–359. doi: 10.1016/s0092-8674(85)80007-6. [DOI] [PubMed] [Google Scholar]
  7. Bringmann P., Lührmann R. Purification of the individual snRNPs U1, U2, U5 and U4/U6 from HeLa cells and characterization of their protein constituents. EMBO J. 1986 Dec 20;5(13):3509–3516. doi: 10.1002/j.1460-2075.1986.tb04676.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brown D. T., Morris G. F., Chodchoy N., Sprecher C., Marzluff W. F. Structure of the sea urchin U1 RNA repeat. Nucleic Acids Res. 1985 Jan 25;13(2):537–556. doi: 10.1093/nar/13.2.537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Castaño J. G., Ornberg R., Koster J. G., Tobian J. A., Zasloff M. Eukaryotic pre-tRNA 5' processing nuclease: copurification with a complex cylindrical particle. Cell. 1986 Aug 1;46(3):377–385. doi: 10.1016/0092-8674(86)90658-6. [DOI] [PubMed] [Google Scholar]
  10. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  11. Driscoll J., Goldberg A. L. Skeletal muscle proteasome can degrade proteins in an ATP-dependent process that does not require ubiquitin. Proc Natl Acad Sci U S A. 1989 Feb;86(3):787–791. doi: 10.1073/pnas.86.3.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Falkenburg P. E., Haass C., Kloetzel P. M., Niedel B., Kopp F., Kuehn L., Dahlmann B. Drosophila small cytoplasmic 19S ribonucleoprotein is homologous to the rat multicatalytic proteinase. Nature. 1988 Jan 14;331(6152):190–192. doi: 10.1038/331190a0. [DOI] [PubMed] [Google Scholar]
  13. Grainger J. L., Winkler M. M. Fertilization triggers unmasking of maternal mRNAs in sea urchin eggs. Mol Cell Biol. 1987 Nov;7(11):3947–3954. doi: 10.1128/mcb.7.11.3947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grainger J. L., von Brunn A., Winkler M. M. Transient synthesis of a specific set of proteins during the rapid cleavage phase of sea urchin development. Dev Biol. 1986 Apr;114(2):403–415. doi: 10.1016/0012-1606(86)90205-8. [DOI] [PubMed] [Google Scholar]
  15. Grossi de Sa M. F., Martins de Sa C., Harper F., Coux O., Akhayat O., Pal J. K., Florentin Y., Scherrer K. Cytolocalization of prosomes as a function of differentiation. J Cell Sci. 1988 Feb;89(Pt 2):151–165. doi: 10.1242/jcs.89.2.151. [DOI] [PubMed] [Google Scholar]
  16. HINEGARDNER R. T. The isolation of nuclei from eggs and embryos of the sea urchin. J Cell Biol. 1962 Dec;15:503–508. doi: 10.1083/jcb.15.3.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hough R., Pratt G., Rechsteiner M. Purification of two high molecular weight proteases from rabbit reticulocyte lysate. J Biol Chem. 1987 Jun 15;262(17):8303–8313. [PubMed] [Google Scholar]
  18. Kari B. E., Rottmann W. L. Analysis of changes in a yolk glycoprotein complex in the developing sea urchin embryo. Dev Biol. 1985 Mar;108(1):18–25. doi: 10.1016/0012-1606(85)90004-1. [DOI] [PubMed] [Google Scholar]
  19. Kloetzel P. M., Falkenburg P. E., Hössl P., Glätzer K. H. The 19S ring-type particles of Drosophila. Cytological and biochemical analysis of their intracellular association and distribution. Exp Cell Res. 1987 May;170(1):204–213. doi: 10.1016/0014-4827(87)90130-3. [DOI] [PubMed] [Google Scholar]
  20. Martins de Sa C., Grossi de Sa M. F., Akhayat O., Broders F., Scherrer K., Horsch A., Schmid H. P. Prosomes. Ubiquity and inter-species structural variation. J Mol Biol. 1986 Feb 20;187(4):479–493. doi: 10.1016/0022-2836(86)90328-1. [DOI] [PubMed] [Google Scholar]
  21. Moon R. T., Danilchik M. V., Hille M. B. An assessment of the masked message hypothesis: sea urchin egg messenger ribonucleoprotein complexes are efficient templates for in vitro protein synthesis. Dev Biol. 1982 Oct;93(2):389–403. doi: 10.1016/0012-1606(82)90126-9. [DOI] [PubMed] [Google Scholar]
  22. Nash M. A., Kozak S. E., Angerer L. M., Angerer R. C., Schatten H., Schatten G., Marzluff W. F. Sea urchin maternal and embryonic U1 RNAs are spatially segregated in early embryos. J Cell Biol. 1987 May;104(5):1133–1142. doi: 10.1083/jcb.104.5.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  24. Pettersson I., Hinterberger M., Mimori T., Gottlieb E., Steitz J. A. The structure of mammalian small nuclear ribonucleoproteins. Identification of multiple protein components reactive with anti-(U1)ribonucleoprotein and anti-Sm autoantibodies. J Biol Chem. 1984 May 10;259(9):5907–5914. [PubMed] [Google Scholar]
  25. Ruzdijic S., Pederson T. Evidence for an association between U1 RNA and interspersed repeat single-copy RNAs in the cytoplasm of sea urchin eggs. Development. 1987 Sep;101(1):107–116. [PubMed] [Google Scholar]
  26. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  27. Schmid H. P., Akhayat O., Martins De Sa C., Puvion F., Koehler K., Scherrer K. The prosome: an ubiquitous morphologically distinct RNP particle associated with repressed mRNPs and containing specific ScRNA and a characteristic set of proteins. EMBO J. 1984 Jan;3(1):29–34. doi: 10.1002/j.1460-2075.1984.tb01757.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tanaka K., Ii K., Ichihara A., Waxman L., Goldberg A. L. A high molecular weight protease in the cytosol of rat liver. I. Purification, enzymological properties, and tissue distribution. J Biol Chem. 1986 Nov 15;261(32):15197–15203. [PubMed] [Google Scholar]
  29. Tanaka K., Yoshimura T., Kumatori A., Ichihara A., Ikai A., Nishigai M., Kameyama K., Takagi T. Proteasomes (multi-protease complexes) as 20 S ring-shaped particles in a variety of eukaryotic cells. J Biol Chem. 1988 Nov 5;263(31):16209–16217. [PubMed] [Google Scholar]
  30. Vincent A., Akhayat O., Goldenberg S., Scherrer K. Differential repression of specific mRNA in erythroblast cytoplasm: a possible role for free mRNP proteins. EMBO J. 1983;2(11):1869–1876. doi: 10.1002/j.1460-2075.1983.tb01673.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wolin S. L., Steitz J. A. The Ro small cytoplasmic ribonucleoproteins: identification of the antigenic protein and its binding site on the Ro RNAs. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1996–2000. doi: 10.1073/pnas.81.7.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES