Mass spectrometric analysis of the anaphase-promoting complex from yeast: Identification of a subunit related to cullins

Science

Volumn 279, Issue 5354, 1998, Pages 1216-1219

  Zachariae, Wolfgang ^a   Shevchenko, Andrej ^b   Andrews, Paul D ^c   Ciosk, Rafael ^a   Galova, Marta ^a   Stark, Michael J R ^c   Mann, Matthias ^b   Nasmyth, Kim ^a  

^a RESEARCH INSTITUTE OF MOLECULAR PATHOLOGY   (Austria)

^b EUROPEAN MOLECULAR BIOLOGY LABORATORY   (Germany)

^c UNIVERSITY OF DUNDEE   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

CULLIN; PROTEIN SUBUNIT; UBIQUITIN PROTEIN LIGASE; UNCLASSIFIED DRUG;

ANAPHASE; ARTICLE; COMPLEX FORMATION; CONTROLLED STUDY; DNA REPLICATION; NONHUMAN; PRIORITY JOURNAL; TANDEM MASS SPECTROMETRY; YEAST;

AMINO ACID SEQUENCE; ANAPHASE; ANIMALS; CELL CYCLE PROTEINS; CULLIN PROTEINS; CYCLINS; DNA REPLICATION; FUNGAL PROTEINS; GENES, FUNGAL; HUMANS; LIGASES; MASS SPECTROMETRY; MITOTIC SPINDLE APPARATUS; MOLECULAR SEQUENCE DATA; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; SEQUENCE ALIGNMENT; UBIQUITIN-PROTEIN LIGASE COMPLEXES; UBIQUITIN-PROTEIN LIGASES; UBIQUITINS;

FUNGI; SACCHAROMYCETALES;

EID: 0032549115     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5354.1216     Document Type: Article

References (39)

1. M. Glotzer, A. W. Murray, M. W. Kirschner, Nature 349, 132 (1991).
2. V. Sudakin et al., Mol. Biol. Cell 6, 185 (1995); R. W. King et al., Cell 81, 279 (1995).
3. V. Sudakin et al., Mol. Biol. Cell 6, 185 (1995); R. W. King et al., Cell 81, 279 (1995).
4. S. Irniger, S. Piatti, C. Michaelis, K. Nasmyth, ibid., p. 269.
5. W. Zachariae and K. Nasmyth, Mol. Biol. Cell 7, 791 (1996).
6. O. Cohen-Fix, J.-M. Peters, M. W. Kirschner, D. Koshland, Genes Dev. 24, 3081 (1996).
7. H. Funabiki et al., Nature 381, 438 (1996).
8. C. Dahman, J. F. X. Diffley, K. Nasmyth, Curr. Biol. 5, 1257 (1995).
9. W. Zachariae, T. H. Shin, M. Galova, B. Obermaier, K. Nasmyth, Science 274, 1201 (1996).
10. J.-M. Peters, R. W. King, C. Höög, M. W. Kirschner, ibid., p. 1199.
11. G. Neubauer et al., Proc. Natl. Acad. Sci. U.S.A. 94, 385 (1997); A. I. Lamond and M. Mann, Trends Cell Biol. 7, 139 (1997).
12. G. Neubauer et al., Proc. Natl. Acad. Sci. U.S.A. 94, 385 (1997); A. I. Lamond and M. Mann, Trends Cell Biol. 7, 139 (1997).
13. 35S-Labeled cells (8) were broken in 0.25 ml of buffer B [50 mM Hepes-KOH (pH 7.3), 5 mM Mg acetate, 0.1% Triton X-100, 20 mM β-glycerophosphate, 10% glycerol, 1 mM NaF, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride pepstatin (1 μg/ ml), proteinase inhibitors (Complete, Boehringer)] containing 60 mM K acetate and bovine serum albumin (BSA, 5 mg/ml). After centrifugation (10 min, 18,000g), extracts were incubated with protein A-Sepharose (0.17 ml) for 30 min and with antibody 9E10 cross-linked to protein A-Sepharose (9E10-beads, 27 μl) for 90 min. Beads were washed three times with 1 ml of buffer B100 (numbers indicate the K acetate concentration in millimolar) containing BSA and with 1 ml of buffers B100, B120, and B150, containing insulin (0.1 mg/ml). Prestained proteins (Rainbow, Amersham) were used as molecular size markers.
14. 10 cells.
15. Proteins were visualized by silver staining and digested in the gel with trypsin [A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Anal. Chem. 68, 850 (1996)]. The digest was analyzed by nanoelectrospray tandem mass spectrometry [M. Wilm et al., Nature 379, 466 (1996)]. Parent ion scans for the immonium ions of leucine and isoleucine were used to detect peptide ions [M. Wilm, G. Neubauer, M. Mann, Anal. Chem. 68, 527 (1996)]. Relative to a BSA standard, the amount of protein available for mass spectrometric identification was ∼10 to 20 ng per band.
16. Proteins were visualized by silver staining and digested in the gel with trypsin [A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Anal. Chem. 68, 850 (1996)]. The digest was analyzed by nanoelectrospray tandem mass spectrometry [M. Wilm et al., Nature 379, 466 (1996)]. Parent ion scans for the immonium ions of leucine and isoleucine were used to detect peptide ions [M. Wilm, G. Neubauer, M. Mann, Anal. Chem. 68, 527 (1996)]. Relative to a BSA standard, the amount of protein available for mass spectrometric identification was ∼10 to 20 ng per band.
17. Proteins were visualized by silver staining and digested in the gel with trypsin [A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Anal. Chem. 68, 850 (1996)]. The digest was analyzed by nanoelectrospray tandem mass spectrometry [M. Wilm et al., Nature 379, 466 (1996)]. Parent ion scans for the immonium ions of leucine and isoleucine were used to detect peptide ions [M. Wilm, G. Neubauer, M. Mann, Anal. Chem. 68, 527 (1996)]. Relative to a BSA standard, the amount of protein available for mass spectrometric identification was ∼10 to 20 ng per band.
18. ORF designations are from the Saccharomyces genome database.
19. A. Shevchenko and M. Mann, unpublished results.
20. + cassette amplified from pFA6a-HIS3MX6 [A. Wach, A. Brachet, C. Alberti-Segui, C. Rebischung, P. Philippsen, Yeast 13, 1065 (1997)].
21. L. H. Hwang and A. W. Murray, Mol. Biol. Cell 8, 1877 (1997).
22. W. Zachariae and K. Nasmyth, unpublished results.
23. Databases were searched with Gapped BLAST [S. F. Altschul et al., Nucleic Acids Res. 25, 3389 (1997)]. ESTs were assembled with AssemblyLIGN (Oxford Molecular, Oxford, UK). Sequences were aligned with CLUSTAL W [J. D. Thompson, D. G. Higgins, T. J. Gibson, Nucleic Acids Res. 22, 4673 (1994)].
24. Databases were searched with Gapped BLAST [S. F. Altschul et al., Nucleic Acids Res. 25, 3389 (1997)]. ESTs were assembled with AssemblyLIGN (Oxford Molecular, Oxford, UK). Sequences were aligned with CLUSTAL W [J. D. Thompson, D. G. Higgins, T. J. Gibson, Nucleic Acids Res. 22, 4673 (1994)].
25. K. L. B. Borden and P. S. Freemont, Curr. Opin. Struct. biol. 6, 395 (1996).
26. H. Yu et al., Science 279, 1219 (1998).
27. E. T. Kipreos et al., Cell 85, 829 (1996); N. Mathias et al., Mol. Cell. Biol. 16, 6634 (1996).
28. E. T. Kipreos et al., Cell 85, 829 (1996); N. Mathias et al., Mol. Cell. Biol. 16, 6634 (1996).
29. D. Skowyra et al., Cell 91, 209 (1997); R. Feldman, C. C. Correll, K. B. Kaplan, R. J. Deshaies, ibid., p. 221.
30. D. Skowyra et al., Cell 91, 209 (1997); R. Feldman, C. C. Correll, K. B. Kaplan, R. J. Deshaies, ibid., p. 221.
31. E. Schwob, T. Böhm, M. D. Mendenhall, K. Nasmyth, ibid. 79, 233 (1994).
32. A. R. Willems et al., ibid. 86, 453 (1996); M. Tyers, personal communication.
33. A. R. Willems et al., ibid. 86, 453 (1996); M. Tyers, personal communication.
34. apc2 mutants were generated as described [S. H. MacKelvie, P. D. Andrews, M. J. R. Stark, Mol. Cell. Biol. 15, 3777 (1995)]. The Xho I to Nde I fragment was removed from the APC2 gene (Cla I to Bgl II) in a CEN-LEU2 plasmid. This DNA was transformed, together with a mutagenized apc2 fragment (-29 to +2554, ATG = +1), into a Δapc2::TRP1 strain containing a CEN-UR43-APC2 plasmid. After selection of transformants showing cell cycle arrest at 37°C, mutant apc2 alleles and the wild-type gene were integrated at the his3 locus of the Δapc2::TRP1 strain.
35. C. Michaelis, R. Ciosk, K. Nasmyth, Cell 91, 35 (1997).
36. 1 cells were elutriated from strains grown at 21°C [E. Schwob and K. Nasmyth, Genes Dey. 7, 1160 (1993)]. Flow cytometric DNA quantitation, indirect immunofluorescence (to detect Pds1-Myc18p and spindles), and observation of tetR-GFP were done as described (27).
37. Amino acids: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
38. M. Mann and M. Wilm, Anal. Chem. 66, 4390 (1994).
39. We thank A. Hyman for bringing together the labs of M.M. and K.N.; A. Schleiffer and B. Habermann for sequence alignments; A. Wach for pFA6a-HIS3MX6; H. Yu, J.-M. Peters, R. King, M. Kirschner, and M. Tyers for communicating results; and M. Glotzer, L. Huber, U. Mühlner, and J.-M. Peters for reading the manuscript.