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The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation.

Wigge PA, Kilmartin JV - J. Cell Biol. (2001)

Bottom Line: Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere.Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells.Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.

ABSTRACT
We have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

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Silver-stained gel of the purified Ndc80p complex, prepared by tagging each of the individual components with prA and affinity purification on an IgG-Sepharose column. Components were identified by MALDI mass spectrometry (see Materials and Methods). Note that in the second lane, Ndc80p and Nuf2p-prA comigrate. The lane marked K699 shows an untagged strain.
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Figure 1: Silver-stained gel of the purified Ndc80p complex, prepared by tagging each of the individual components with prA and affinity purification on an IgG-Sepharose column. Components were identified by MALDI mass spectrometry (see Materials and Methods). Note that in the second lane, Ndc80p and Nuf2p-prA comigrate. The lane marked K699 shows an untagged strain.

Mentions: Proteins in SDS gel bands were identified by mass spectrometry after increasing the loading 10-fold so that the silver-stained bands in Fig. 1 were just visible by Coomassie staining. Gel bands were digested with trypsin (Wigge et al. 1998) and the tryptic peptide masses were determined by MALDI mass spectrometry in a PerSeptive Biosystems Voyager-DE STR mass spectrometer using external standards or matrix peaks and trypsin peptides as internal standards. The National Center for Biotechnology Information nonredundant database of about half a million proteins was searched using MS-fit (available at http://prospector.ucsf.edu) set at 50 parts per million, 0–300 kD. All of the proteins identified in Fig. 1 were the top match with MOWSE (Pappin et al. 1993) scores (P factor 0.4) of between 3 × 105 and 4 × 1017 apart from the Spc25p–prA track where Spc24p and prA had scores of 9 × 104 and 2 × 103. A second search allowing methionine oxidation, protein NH2-terminal acetylation, and two missed tryptic cleavages was then carried out to match further peptides. For the identifications in this paper the first number in brackets is the number of tryptic peptides identified followed by the percentage of sequence covered; these were (going from left to right across the gel in Fig. 1 for each individual protein) Ndc80p: 19, 38% (vector: 9, 43%); 23, 39% (second track); 30, 46% (third track); 28, 47% (fourth track); Nuf2p: 20, 45%; 13, 34% (vector: 6, 36%); 14, 37%; 16, 42%; Spc24p: 13, 58%; 9, 55%; 13, 66% (vector: 10, 46%); 13, 69%; Spc25p: 7, 33%; 11, 59%; 12, 60%; 10, 49% (vector: 8, 37%); and prA: 8, 35%.


The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation.

Wigge PA, Kilmartin JV - J. Cell Biol. (2001)

Silver-stained gel of the purified Ndc80p complex, prepared by tagging each of the individual components with prA and affinity purification on an IgG-Sepharose column. Components were identified by MALDI mass spectrometry (see Materials and Methods). Note that in the second lane, Ndc80p and Nuf2p-prA comigrate. The lane marked K699 shows an untagged strain.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199619&req=5

Figure 1: Silver-stained gel of the purified Ndc80p complex, prepared by tagging each of the individual components with prA and affinity purification on an IgG-Sepharose column. Components were identified by MALDI mass spectrometry (see Materials and Methods). Note that in the second lane, Ndc80p and Nuf2p-prA comigrate. The lane marked K699 shows an untagged strain.
Mentions: Proteins in SDS gel bands were identified by mass spectrometry after increasing the loading 10-fold so that the silver-stained bands in Fig. 1 were just visible by Coomassie staining. Gel bands were digested with trypsin (Wigge et al. 1998) and the tryptic peptide masses were determined by MALDI mass spectrometry in a PerSeptive Biosystems Voyager-DE STR mass spectrometer using external standards or matrix peaks and trypsin peptides as internal standards. The National Center for Biotechnology Information nonredundant database of about half a million proteins was searched using MS-fit (available at http://prospector.ucsf.edu) set at 50 parts per million, 0–300 kD. All of the proteins identified in Fig. 1 were the top match with MOWSE (Pappin et al. 1993) scores (P factor 0.4) of between 3 × 105 and 4 × 1017 apart from the Spc25p–prA track where Spc24p and prA had scores of 9 × 104 and 2 × 103. A second search allowing methionine oxidation, protein NH2-terminal acetylation, and two missed tryptic cleavages was then carried out to match further peptides. For the identifications in this paper the first number in brackets is the number of tryptic peptides identified followed by the percentage of sequence covered; these were (going from left to right across the gel in Fig. 1 for each individual protein) Ndc80p: 19, 38% (vector: 9, 43%); 23, 39% (second track); 30, 46% (third track); 28, 47% (fourth track); Nuf2p: 20, 45%; 13, 34% (vector: 6, 36%); 14, 37%; 16, 42%; Spc24p: 13, 58%; 9, 55%; 13, 66% (vector: 10, 46%); 13, 69%; Spc25p: 7, 33%; 11, 59%; 12, 60%; 10, 49% (vector: 8, 37%); and prA: 8, 35%.

Bottom Line: Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere.Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells.Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.

ABSTRACT
We have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

Show MeSH
Related in: MedlinePlus