Identification of subunits of the anaphase-promoting complex of Saccharomyces cerevisiae

Science

Volumn 274, Issue 5290, 1996, Pages 1201-1204

  Zachariae, Wolfgang ^a   Shin, Tae Ho ^a,c   Galova, Marta ^a   Obermaier, Brigitte ^b   Nasmyth, Kim ^a  

^a RESEARCH INSTITUTE OF MOLECULAR PATHOLOGY   (Austria)

^b MediGene AG   (Germany)

^c UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CYCLIN B; PROTEIN SUBUNIT; UBIQUITIN PROTEIN LIGASE;

ANAPHASE; ARTICLE; MITOSIS; PRIORITY JOURNAL; PROTEIN DEGRADATION; PROTEIN QUATERNARY STRUCTURE; SACCHAROMYCES CEREVISIAE;

ANAPHASE; CELL CYCLE PROTEINS; CELL DIVISION; CENTRIFUGATION, DENSITY GRADIENT; CYCLINS; FLUORESCENT ANTIBODY TECHNIQUE, INDIRECT; FUNGAL PROTEINS; G1 PHASE; GENES, FUNGAL; LIGASES; MITOSIS; SACCHAROMYCES CEREVISIAE; UBIQUITIN-PROTEIN LIGASES; UBIQUITINS;

ASPERGILLUS; EMERICELLA NIDULANS; FUNGI; SACCHAROMYCES CEREVISIAE; SACCHAROMYCETALES; VERTEBRATA;

EID: 0343829343     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5290.1201     Document Type: Article

References (39)

1. A. W. Murray, M. J. Solomon, M. W. Kirschner, Nature 339, 503 (1989); F. C: Luca, E. K. Shibuya, C. E. Dohrman, J. V. Ruderman. EMBO J. 10, 4311 (1991).
2. A. W. Murray, M. J. Solomon, M. W. Kirschner, Nature 339, 503 (1989); F. C: Luca, E. K. Shibuya, C. E. Dohrman, J. V. Ruderman. EMBO J. 10, 4311 (1991).
3. U. Surana et al., EMBO J. 12, 1969 (1993).
4. C. Dahman, J. F. X. Diffiey, K. Nasmyth, Curr. Biol. 5, 1257 (1095).
5. R. W. King et al., Cell 81, 279 (1995).
6. V. Sudakin et al., Mol. Biol. Cell 6, 185 (1995).
7. A. Ciechanover, Cell 79, 13 (1994); J. M. Peters, Trends Biochem. Sci. 19, 377 (1994).
8. A. Ciechanover, Cell 79, 13 (1994); J. M. Peters, Trends Biochem. Sci. 19, 377 (1994).
9. S. Irniger, S. Piatti, C. Michaelis, K. Nasmyth, Cell 81, 269 (1995).
10. W. Zachariae and K. Nasmyth, Mol. Biol. Cell 7, 791 (1996).
11. T. Hirano, Y. Hiraoka, M. Yanagida, J. Cell Biol. 106, 1171 (1988); K. L. O'Donnell, A. H. Osmani, S. A. Osmani, N. R. Morris, J. Cell Sci. 99, 711 (1991); I. Samejima and M. Yanagida, J. Cell Biol. 127, 1655 (1994); S. Tugendreich, J. Tomkiel, W. Earnshaw, P. Hieter, Cell 81, 261 (1995).
12. T. Hirano, Y. Hiraoka, M. Yanagida, J. Cell Biol. 106, 1171 (1988); K. L. O'Donnell, A. H. Osmani, S. A. Osmani, N. R. Morris, J. Cell Sci. 99, 711 (1991); I. Samejima and M. Yanagida, J. Cell Biol. 127, 1655 (1994); S. Tugendreich, J. Tomkiel, W. Earnshaw, P. Hieter, Cell 81, 261 (1995).
13. T. Hirano, Y. Hiraoka, M. Yanagida, J. Cell Biol. 106, 1171 (1988); K. L. O'Donnell, A. H. Osmani, S. A. Osmani, N. R. Morris, J. Cell Sci. 99, 711 (1991); I. Samejima and M. Yanagida, J. Cell Biol. 127, 1655 (1994); S. Tugendreich, J. Tomkiel, W. Earnshaw, P. Hieter, Cell 81, 261 (1995).
14. T. Hirano, Y. Hiraoka, M. Yanagida, J. Cell Biol. 106, 1171 (1988); K. L. O'Donnell, A. H. Osmani, S. A. Osmani, N. R. Morris, J. Cell Sci. 99, 711 (1991); I. Samejima and M. Yanagida, J. Cell Biol. 127, 1655 (1994); S. Tugendreich, J. Tomkiel, W. Earnshaw, P. Hieter, Cell 81, 261 (1995).
15. J. R. Lamb, W. A. Michaud, R. S. Sikorski, P. A. Hieter, EMBO J. 13, 4321 (1994).
16. S. L. Holloway, M. Glotzer, R. W. King, A. W. Murray, Cell 73, 1393 (1993).
17. H. Funabiki et al., Nature 381, 438 (1996).
18. A. Amon, S. Irniger, K. Nasmyth, Cell 77, 1037 (1994).
19. 1-arrested cells were screened for accumulation of Clb2-lacZp at 37°C (7). Mutants were further analyzed that arrested as large, budded cells with a 2C DNA content after cycling cultures were shifted from 25° to 37°C. The original mutants were backcrossed at least three times to wild type.
20. Mutants were transformed with a genomic library [F. Cvrckova and K. Nasmyth, EMBO J. 12, 5277 (1993)], and plasmids allowing growth at 37°C were recovered. The ape1-1 mutation was complemented by the open reading frame YNL172W on chromosome XIV (Saccharomyces Genome Database). The cdc26-519 and apc1-1 mutants were crossed to strains containing a CDC26::LEU2 or an APC1::HIS3 fusion, respectively. All tetrads were parental ditypes.
21. H. Akari, K. Awane, N. Ogawa, Y. Oshima, Mol. Gen. Genet. 231, 329 (1992).
22. + spores that were viable at 25°C but arrested as large, budded cells at 37°C and two Ura spores that grew normally at 37°C.
23. W. Zachariae and K. Nasmyth, unpublished data.
24. D. B. Engle et al., J. Biol. Chem. 265, 16132 (1990).
25. M. Starborg, E. Brundell, K. Gell, C. Höög, ibid. 269, 24133 (1994).
26. T. H. Shin and K. Nasmyth, unpublished results.
27. A 4.3-kb Bam HI to Sty I fragment was replaced by HIS3, and the apc1::HIS3 construct was used to disrupt one APC1 allele in a diploid strain.
28. Two hundred base pairs (bp) of coding region with several HA or Myc epitope tags inserted in front of the stop codon and 200 bp of 3′-noncoding sequence were cloned into an integrative vector. The resulting plasmid was integrated at the respective genomic locus. Strains expressing two epitopetagged proteins were obtained by genetic crosses. At 37°C all strains, including a clb 1Δ CLB2-myc12 strain, grew normally, demonstrating that the epitope-tagged proteins were fully functional.
29. 35S-cysteine in 1.5 ml of medium for 2 hours at 30°C. Cells were broken in 0.25 ml of buffer B [buffer A (8) diluted 1:3] containing 1.5 mg of unlabeled protein from a pep4 strain (K5517) (8). After centrifugation (10 min, 12,000g), extracts were sequentially incubated with protein A-Sepharose (160 μl) and antibody 9E10 cross-linked to protein A-Sepharose (25 μl). The beads were washed with buffer B (4 × 1 ml), buffer B with 120 mM K acetate (1 ml), and buffer B with 150 mM K acetate (1 ml). Bound proteins were separated on SDS-polyacrylamide gels and detected by fluorography.
30. Cells were grown in YEP medium with 2% raffinose. Extracts (2 mg of protein) prepared in buffer C [buffer A (8) without glycerol, diluted 1:3] were layered on 10 to 35% glycerol gradients in buffer C and centrifuged for 15 hours at 28,000 rpm in a Beckman SW40 rotor.
31. Cells were prepared for indirect immunofluorescence [K. Nasmyth, G. Adolf, D. Lydall, A. Seddon, Cell 62, 631 (1990)], and pictures were taken on Kodak T-MAX400 film or with a charge-coupled device camera mounted on a Zeiss Axiophot microscope. Myc-tagged proteins were detected with the 9E10 antibody and a CY3-conjugated secondary antibody. Spindles were detected with a rabbit antiserum to yeast tubulin and a fluorescein isothiocyanate-conjugated secondary antibody.
32. R. Ciosk and K. Nasmyth, unpublished results.
33. Strains were grown in SC medium [F. Sherman, Methods Enzymol. 194, 3 (1991)] containing 2% raffinose at 25°C, Small, unbudded cells were isolated by centrifugal elutriation [E. Schwob and K. Nasmyth, Genes Des. 7, 1160 (1993)] and inoculated into YEP medium with 2% glucose at 37°C. Clb2-associated histone H1 kinase (2) was quantified with a Phosphorlmager (Molecular Dynamics). A FACScan (Becton-Dickinson) was used for analysis of cellular DNA content [C. B. Epstein and F. R. Cross, Genes Dev. 6, 1695 (1992)].
34. Strains were grown in SC medium [F. Sherman, Methods Enzymol. 194, 3 (1991)] containing 2% raffinose at 25°C, Small, unbudded cells were isolated by centrifugal elutriation [E. Schwob and K. Nasmyth, Genes Des. 7, 1160 (1993)] and inoculated into YEP medium with 2% glucose at 37°C. Clb2-associated histone H1 kinase (2) was quantified with a Phosphorlmager (Molecular Dynamics). A FACScan (Becton-Dickinson) was used for analysis of cellular DNA content [C. B. Epstein and F. R. Cross, Genes Dev. 6, 1695 (1992)].
35. Strains were grown in SC medium [F. Sherman, Methods Enzymol. 194, 3 (1991)] containing 2% raffinose at 25°C, Small, unbudded cells were isolated by centrifugal elutriation [E. Schwob and K. Nasmyth, Genes Des. 7, 1160 (1993)] and inoculated into YEP medium with 2% glucose at 37°C. Clb2-associated histone H1 kinase (2) was quantified with a Phosphorlmager (Molecular Dynamics). A FACScan (Becton-Dickinson) was used for analysis of cellular DNA content [C. B. Epstein and F. R. Cross, Genes Dev. 6, 1695 (1992)].
36. J. M. Peters, R. W. King, C. Höög, M. W. Kirschner, Science 274, 1199 (1996).
37. S. A. Osmani, D. B. Engle, J. H. Doonan, N. R. Morris, Cell 52, 241 (1988); S. W. James, P. M. Mirabito, P. C. Scacheri, N. R. Morris, J. Cell Sci. 108, 3485 (1995).
38. S. A. Osmani, D. B. Engle, J. H. Doonan, N. R. Morris, Cell 52, 241 (1988); S. W. James, P. M. Mirabito, P. C. Scacheri, N. R. Morris, J. Cell Sci. 108, 3485 (1995).
39. We thank R. Ciesk for tagging of CSE1; C. Michaelis, K. Breunig, and S. Irniger for DNAs and strains; M. Oft for help in fluorescence microscopy and computer graphics; J. Dohmen and R. Egner for antisera; G. Lamm, T. Jenuwein, and T. Czerny for reading the manuscript; and H. Tkadletz for photographs. W.Z. was supported by a European Molecular Biology Organization long-term fellowship.