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Implication of a novel multiprotein Dam1p complex in outer kinetochore function.

Cheeseman IM, Brew C, Wolyniak M, Desai A, Anderson S, Muster N, Yates JR, Huffaker TC, Drubin DG, Barnes G - J. Cell Biol. (2001)

Bottom Line: We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of approximately 0.5 microM.To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19-green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1.These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

ABSTRACT
Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an approximately 245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of approximately 0.5 microM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19-green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

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dad2 and ask1 mutants show spindle defects. (A) Immunoblots of ask1td and dad2td strains showing degradation of the fusion protein. Degron-tagged alleles of ask1 and dad2 were grown at 25°C and shifted to 37°C at t = 0 h Protein samples were immunoblotted with anti-HA antibodies to detect the DHFRts–HA fusion protein. (B) ask1td and dad2td mutant phenotypes. Degron-tagged alleles of ask1 and dad2 were grown at 25°C and shifted to 37°C for 3 h. They were then processed for tubulin immunofluorescence and DNA staining (DAPI). At this time point, 90% of large budded ask1td and 82% of large budded dad2td cells showed short spindle structures and a single mass of DNA, whereas 10% of ask1td and 15% of dad2td cells showed broken down spindles (n = 100 cells/sample). Bar, 5 μm.
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fig4: dad2 and ask1 mutants show spindle defects. (A) Immunoblots of ask1td and dad2td strains showing degradation of the fusion protein. Degron-tagged alleles of ask1 and dad2 were grown at 25°C and shifted to 37°C at t = 0 h Protein samples were immunoblotted with anti-HA antibodies to detect the DHFRts–HA fusion protein. (B) ask1td and dad2td mutant phenotypes. Degron-tagged alleles of ask1 and dad2 were grown at 25°C and shifted to 37°C for 3 h. They were then processed for tubulin immunofluorescence and DNA staining (DAPI). At this time point, 90% of large budded ask1td and 82% of large budded dad2td cells showed short spindle structures and a single mass of DNA, whereas 10% of ask1td and 15% of dad2td cells showed broken down spindles (n = 100 cells/sample). Bar, 5 μm.

Mentions: Previous analyses of temperature-sensitive duo1, dam1, and dad1 mutants revealed a range of defects in spindle integrity (Hofmann et al., 1998; Cheeseman et al., 2001; Enquist-Newman et al., 2001). If the newly identified subunits are functionally relevant, we predicted that they would show similar mutant phenotypes. SPC19, SPC34, DAD2, and ASK1 are all essential genes (Wigge et al., 1998; Winzeler et al., 1999). To determine the loss of function phenotypes of some of these additional subunits, we generated degron-tagged alleles of ask1 and dad2 (termed td, for temperature-degron [Dohmen et al., 1994]). When ask1td and dad2td mutants were shifted to the restrictive temperature, the Ask1p and Dad2p fusion proteins were targeted for degradation (Fig. 4 A), and the mutants arrested with a high proportion of large-budded cells. When the mutant spindles were examined, we found that the majority of ask1td and dad2td cells arrested with a short mitotic spindle and a single mass of DNA (Fig. 4 B) similar to what we have described previously for duo1-2, dam1-9, and dad1-1 mutants. A smaller percentage of ask1td and dad2td mutant cells showed spindles that had broken down partially or completely in the middle and elongated beyond the short spindle stage (Fig. 4 B, insets). In total, these results provide strong phenotypic evidence that Ask1p and Dad2p function as components of the Dam1p complex to maintain spindle integrity.


Implication of a novel multiprotein Dam1p complex in outer kinetochore function.

Cheeseman IM, Brew C, Wolyniak M, Desai A, Anderson S, Muster N, Yates JR, Huffaker TC, Drubin DG, Barnes G - J. Cell Biol. (2001)

dad2 and ask1 mutants show spindle defects. (A) Immunoblots of ask1td and dad2td strains showing degradation of the fusion protein. Degron-tagged alleles of ask1 and dad2 were grown at 25°C and shifted to 37°C at t = 0 h Protein samples were immunoblotted with anti-HA antibodies to detect the DHFRts–HA fusion protein. (B) ask1td and dad2td mutant phenotypes. Degron-tagged alleles of ask1 and dad2 were grown at 25°C and shifted to 37°C for 3 h. They were then processed for tubulin immunofluorescence and DNA staining (DAPI). At this time point, 90% of large budded ask1td and 82% of large budded dad2td cells showed short spindle structures and a single mass of DNA, whereas 10% of ask1td and 15% of dad2td cells showed broken down spindles (n = 100 cells/sample). Bar, 5 μm.
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fig4: dad2 and ask1 mutants show spindle defects. (A) Immunoblots of ask1td and dad2td strains showing degradation of the fusion protein. Degron-tagged alleles of ask1 and dad2 were grown at 25°C and shifted to 37°C at t = 0 h Protein samples were immunoblotted with anti-HA antibodies to detect the DHFRts–HA fusion protein. (B) ask1td and dad2td mutant phenotypes. Degron-tagged alleles of ask1 and dad2 were grown at 25°C and shifted to 37°C for 3 h. They were then processed for tubulin immunofluorescence and DNA staining (DAPI). At this time point, 90% of large budded ask1td and 82% of large budded dad2td cells showed short spindle structures and a single mass of DNA, whereas 10% of ask1td and 15% of dad2td cells showed broken down spindles (n = 100 cells/sample). Bar, 5 μm.
Mentions: Previous analyses of temperature-sensitive duo1, dam1, and dad1 mutants revealed a range of defects in spindle integrity (Hofmann et al., 1998; Cheeseman et al., 2001; Enquist-Newman et al., 2001). If the newly identified subunits are functionally relevant, we predicted that they would show similar mutant phenotypes. SPC19, SPC34, DAD2, and ASK1 are all essential genes (Wigge et al., 1998; Winzeler et al., 1999). To determine the loss of function phenotypes of some of these additional subunits, we generated degron-tagged alleles of ask1 and dad2 (termed td, for temperature-degron [Dohmen et al., 1994]). When ask1td and dad2td mutants were shifted to the restrictive temperature, the Ask1p and Dad2p fusion proteins were targeted for degradation (Fig. 4 A), and the mutants arrested with a high proportion of large-budded cells. When the mutant spindles were examined, we found that the majority of ask1td and dad2td cells arrested with a short mitotic spindle and a single mass of DNA (Fig. 4 B) similar to what we have described previously for duo1-2, dam1-9, and dad1-1 mutants. A smaller percentage of ask1td and dad2td mutant cells showed spindles that had broken down partially or completely in the middle and elongated beyond the short spindle stage (Fig. 4 B, insets). In total, these results provide strong phenotypic evidence that Ask1p and Dad2p function as components of the Dam1p complex to maintain spindle integrity.

Bottom Line: We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of approximately 0.5 microM.To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19-green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1.These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

ABSTRACT
Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an approximately 245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of approximately 0.5 microM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19-green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

Show MeSH
Related in: MedlinePlus