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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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Purified Dam1p complex binds to microtubules directly with ∼0.5 μM affinity. Purified Dam1p complex (∼5–10 nM) was incubated with varying concentrations of microtubules, which were then pelleted by centrifugation. The amount of complex bound to microtubules was determined by quantifying Duo1p and Dam1p in the pellet and supernatant fractions. (A) Percentage of protein bound to microtubules plotted with respect to the concentration of microtubules in the reaction. (B) Western blots showing the amount of Dam1p or Duo1p that is unbound (S, supernatant) or bound (P, pellet) at each concentration of microtubules.
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fig2: Purified Dam1p complex binds to microtubules directly with ∼0.5 μM affinity. Purified Dam1p complex (∼5–10 nM) was incubated with varying concentrations of microtubules, which were then pelleted by centrifugation. The amount of complex bound to microtubules was determined by quantifying Duo1p and Dam1p in the pellet and supernatant fractions. (A) Percentage of protein bound to microtubules plotted with respect to the concentration of microtubules in the reaction. (B) Western blots showing the amount of Dam1p or Duo1p that is unbound (S, supernatant) or bound (P, pellet) at each concentration of microtubules.

Mentions: Since a Dam1p in vitro translation product was shown previously to bind to microtubules directly (Hofmann et al., 1998), this suggested that the entire Dam1p complex may also bind to microtubules. To test this possibility, we incubated limiting amounts of purified Dam1p complex (∼5–10 nM) in the presence of varying concentrations of taxol-stabilized bovine brain microtubules (0–5 μM). We then subjected these reactions to ultracentrifugation to determine the percentage of Dam1p complex bound to each concentration of microtubules (determined by examining Duo1p and Dam1p). We determined that the Dam1p complex binds directly to microtubules in vitro with an affinity of ∼0.5 μM (Fig. 2). This value is consistent with the affinity that we determined previously for the binding of in vitro–translated Dam1p (1 μM [Hofmann et al., 1998]) or Escherichia coli–purified Dam1p (∼0.5 μM; unpublished data) to microtubules. Interestingly, we found that both phosphorylated and unphosphorylated forms of Dam1p complex bind to microtubules with similar affinities (unpublished data), suggesting that protein phosphorylation of the Dam1p complex regulates an activity other than microtubule binding.


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)

Purified Dam1p complex binds to microtubules directly with ∼0.5 μM affinity. Purified Dam1p complex (∼5–10 nM) was incubated with varying concentrations of microtubules, which were then pelleted by centrifugation. The amount of complex bound to microtubules was determined by quantifying Duo1p and Dam1p in the pellet and supernatant fractions. (A) Percentage of protein bound to microtubules plotted with respect to the concentration of microtubules in the reaction. (B) Western blots showing the amount of Dam1p or Duo1p that is unbound (S, supernatant) or bound (P, pellet) at each concentration of microtubules.
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Related In: Results  -  Collection

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fig2: Purified Dam1p complex binds to microtubules directly with ∼0.5 μM affinity. Purified Dam1p complex (∼5–10 nM) was incubated with varying concentrations of microtubules, which were then pelleted by centrifugation. The amount of complex bound to microtubules was determined by quantifying Duo1p and Dam1p in the pellet and supernatant fractions. (A) Percentage of protein bound to microtubules plotted with respect to the concentration of microtubules in the reaction. (B) Western blots showing the amount of Dam1p or Duo1p that is unbound (S, supernatant) or bound (P, pellet) at each concentration of microtubules.
Mentions: Since a Dam1p in vitro translation product was shown previously to bind to microtubules directly (Hofmann et al., 1998), this suggested that the entire Dam1p complex may also bind to microtubules. To test this possibility, we incubated limiting amounts of purified Dam1p complex (∼5–10 nM) in the presence of varying concentrations of taxol-stabilized bovine brain microtubules (0–5 μM). We then subjected these reactions to ultracentrifugation to determine the percentage of Dam1p complex bound to each concentration of microtubules (determined by examining Duo1p and Dam1p). We determined that the Dam1p complex binds directly to microtubules in vitro with an affinity of ∼0.5 μM (Fig. 2). This value is consistent with the affinity that we determined previously for the binding of in vitro–translated Dam1p (1 μM [Hofmann et al., 1998]) or Escherichia coli–purified Dam1p (∼0.5 μM; unpublished data) to microtubules. Interestingly, we found that both phosphorylated and unphosphorylated forms of Dam1p complex bind to microtubules with similar affinities (unpublished data), suggesting that protein phosphorylation of the Dam1p complex regulates an activity other than microtubule binding.

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