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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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Spc19p, Spc34p, Dad2p, and Ask1p localize to spindles and kinetochores. (A) GFP fluorescence and corresponding DIC images showing the localization of the indicated fusion protein to the mitotic spindle. (B) Cells expressing the indicated GFP fusion proteins were prepared for chromosome spreads as described (Loidl et al., 1998). They were then processed for immunofluorescence and stained with anti-GFP and anti-Dam1p antibodies. Bar, 5 μm.
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fig3: Spc19p, Spc34p, Dad2p, and Ask1p localize to spindles and kinetochores. (A) GFP fluorescence and corresponding DIC images showing the localization of the indicated fusion protein to the mitotic spindle. (B) Cells expressing the indicated GFP fusion proteins were prepared for chromosome spreads as described (Loidl et al., 1998). They were then processed for immunofluorescence and stained with anti-GFP and anti-Dam1p antibodies. Bar, 5 μm.

Mentions: The purification described above isolated a tight complex of proteins that behaved as a functional unit in vitro with respect to binding to microtubules. We next wanted to establish whether these proteins formed a functionally relevant complex in vivo. Based on our previous analyses of Duo1p, Dam1p, and Dad1p (Hofmann et al., 1998; Cheeseman et al., 2001; Enquist-Newman et al., 2001), we predicted that the newly identified subunits would localize to the mitotic spindle. In fact, COOH-terminal fusions between Spc34p, Spc19p, Dad2p, or Ask1p, and GFP all localized to spindle poles and along the length of both short and long mitotic spindles (Fig. 3 A). Interestingly, Dad2-GFP showed strong punctate localization along longer spindles in addition to a weaker uniform straining. Colocalization with tubulin confirmed the localization of these proteins to the spindle but not to cytoplasmic microtubules (unpublished data). GFP fusions of Spc19p and Spc34p have also been localized along the mitotic spindle by immunofluorescence and immunoelectron microscopy (Wigge et al., 1998).


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)

Spc19p, Spc34p, Dad2p, and Ask1p localize to spindles and kinetochores. (A) GFP fluorescence and corresponding DIC images showing the localization of the indicated fusion protein to the mitotic spindle. (B) Cells expressing the indicated GFP fusion proteins were prepared for chromosome spreads as described (Loidl et al., 1998). They were then processed for immunofluorescence and stained with anti-GFP and anti-Dam1p antibodies. Bar, 5 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199314&req=5

fig3: Spc19p, Spc34p, Dad2p, and Ask1p localize to spindles and kinetochores. (A) GFP fluorescence and corresponding DIC images showing the localization of the indicated fusion protein to the mitotic spindle. (B) Cells expressing the indicated GFP fusion proteins were prepared for chromosome spreads as described (Loidl et al., 1998). They were then processed for immunofluorescence and stained with anti-GFP and anti-Dam1p antibodies. Bar, 5 μm.
Mentions: The purification described above isolated a tight complex of proteins that behaved as a functional unit in vitro with respect to binding to microtubules. We next wanted to establish whether these proteins formed a functionally relevant complex in vivo. Based on our previous analyses of Duo1p, Dam1p, and Dad1p (Hofmann et al., 1998; Cheeseman et al., 2001; Enquist-Newman et al., 2001), we predicted that the newly identified subunits would localize to the mitotic spindle. In fact, COOH-terminal fusions between Spc34p, Spc19p, Dad2p, or Ask1p, and GFP all localized to spindle poles and along the length of both short and long mitotic spindles (Fig. 3 A). Interestingly, Dad2-GFP showed strong punctate localization along longer spindles in addition to a weaker uniform straining. Colocalization with tubulin confirmed the localization of these proteins to the spindle but not to cytoplasmic microtubules (unpublished data). GFP fusions of Spc19p and Spc34p have also been localized along the mitotic spindle by immunofluorescence and immunoelectron microscopy (Wigge et al., 1998).

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