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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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(a) Homologues of S. cerevisiae (Sc) Nuf2p in S. pombe (Sp), C. elegans (Ce), mice (Mm), and humans (Hs), showing homology in the mainly noncoiled coil NH2-terminal region. (b) In four of the homologues there is a break in the coiled coil at around residue 250 associated with an SPEKLK motif. (c) There is a broadly similar distribution of coiled coil domains in the COOH-terminal region of all four homologues. (d) Homologues of Spc24p in S. cerevisiae and S. pombe showing homology in the COOH-terminal region and a similar coiled coil distribution (e).
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Figure 7: (a) Homologues of S. cerevisiae (Sc) Nuf2p in S. pombe (Sp), C. elegans (Ce), mice (Mm), and humans (Hs), showing homology in the mainly noncoiled coil NH2-terminal region. (b) In four of the homologues there is a break in the coiled coil at around residue 250 associated with an SPEKLK motif. (c) There is a broadly similar distribution of coiled coil domains in the COOH-terminal region of all four homologues. (d) Homologues of Spc24p in S. cerevisiae and S. pombe showing homology in the COOH-terminal region and a similar coiled coil distribution (e).

Mentions: Ndc80p has both an S. pombe and a human homologue (Wigge et al. 1998; Zheng et al. 1999), and we also found S. pombe homologues of both Nuf2p and Spc24p (Fig. 7, a and d). Nuf2p and Spc24p are both coiled-coil proteins, and their S. pombe homologues have a similar pattern of coiled-coil domains (Fig. 7c and Fig. e) and in particular homology outside the coiled-coil region (Fig. 7, a and d). We call these S. pombe homologues SpNdc80, SpNuf2, and SpSpc24. We also found a human homologue of Nuf2p. We restricted the BLAST (Altschul et al. 1990) search to the Homo sapiens EST database, increased the E value to 100, and used the most conserved part of the NH2 terminus between S. cerevisiae and S. pombe (residues 79–130 in S. pombe) to avoid matches with other coiled-coil proteins. The top match (Image 1626830) had a low score (E value 13) but did encode some of the residues conserved between the two yeasts. The EST was sequenced and used to match more ESTs (refer to Materials and Methods) to give a sequence encoding a protein (Fig. 7 a) from chromosome I which we call hNuf2R (human Nuf2p-related). A mouse homologue was also identified (Fig. 7 a) which was 73% identical to hNuf2R and one amino acid shorter. When hNuf2R was used to search the nonredundant sequence database using FastA (Pearson and Lipman 1988), the top three matches (Fig. 7 a) were S. pombe Nuf2 (E value 2 × 10−9), S. cerevisiae Nuf2p (1.7 × 10−6), and a Caenorhabditis elegans protein R12B2.4 (7 × 10−6). All of the proteins were similar in length and had a coiled-coil region in the COOH-terminal half (Fig. 7 c), and in addition the S. pombe, C. elegans, mouse and human proteins had a break in the coiled-coil at about residue 250 associated with a SPEKLK motif (Fig. 7 b).


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)

(a) Homologues of S. cerevisiae (Sc) Nuf2p in S. pombe (Sp), C. elegans (Ce), mice (Mm), and humans (Hs), showing homology in the mainly noncoiled coil NH2-terminal region. (b) In four of the homologues there is a break in the coiled coil at around residue 250 associated with an SPEKLK motif. (c) There is a broadly similar distribution of coiled coil domains in the COOH-terminal region of all four homologues. (d) Homologues of Spc24p in S. cerevisiae and S. pombe showing homology in the COOH-terminal region and a similar coiled coil distribution (e).
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Figure 7: (a) Homologues of S. cerevisiae (Sc) Nuf2p in S. pombe (Sp), C. elegans (Ce), mice (Mm), and humans (Hs), showing homology in the mainly noncoiled coil NH2-terminal region. (b) In four of the homologues there is a break in the coiled coil at around residue 250 associated with an SPEKLK motif. (c) There is a broadly similar distribution of coiled coil domains in the COOH-terminal region of all four homologues. (d) Homologues of Spc24p in S. cerevisiae and S. pombe showing homology in the COOH-terminal region and a similar coiled coil distribution (e).
Mentions: Ndc80p has both an S. pombe and a human homologue (Wigge et al. 1998; Zheng et al. 1999), and we also found S. pombe homologues of both Nuf2p and Spc24p (Fig. 7, a and d). Nuf2p and Spc24p are both coiled-coil proteins, and their S. pombe homologues have a similar pattern of coiled-coil domains (Fig. 7c and Fig. e) and in particular homology outside the coiled-coil region (Fig. 7, a and d). We call these S. pombe homologues SpNdc80, SpNuf2, and SpSpc24. We also found a human homologue of Nuf2p. We restricted the BLAST (Altschul et al. 1990) search to the Homo sapiens EST database, increased the E value to 100, and used the most conserved part of the NH2 terminus between S. cerevisiae and S. pombe (residues 79–130 in S. pombe) to avoid matches with other coiled-coil proteins. The top match (Image 1626830) had a low score (E value 13) but did encode some of the residues conserved between the two yeasts. The EST was sequenced and used to match more ESTs (refer to Materials and Methods) to give a sequence encoding a protein (Fig. 7 a) from chromosome I which we call hNuf2R (human Nuf2p-related). A mouse homologue was also identified (Fig. 7 a) which was 73% identical to hNuf2R and one amino acid shorter. When hNuf2R was used to search the nonredundant sequence database using FastA (Pearson and Lipman 1988), the top three matches (Fig. 7 a) were S. pombe Nuf2 (E value 2 × 10−9), S. cerevisiae Nuf2p (1.7 × 10−6), and a Caenorhabditis elegans protein R12B2.4 (7 × 10−6). All of the proteins were similar in length and had a coiled-coil region in the COOH-terminal half (Fig. 7 c), and in addition the S. pombe, C. elegans, mouse and human proteins had a break in the coiled-coil at about residue 250 associated with a SPEKLK motif (Fig. 7 b).

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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