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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.

Show MeSH
Protein interactions establishing the connectivity between spindle microtubules and centromeric DNA. Physical interactions are indicated by lines between proteins. Shapes indicate distinct complexes within the kinetochore. Proteins that interact directly with centromeric DNA are shown in blue. Proteins whose loss of function results in genetic interactions with dam1-1 are shown in red. Protein interaction data are from Chen et al. (1998), Ghosh et al. (2001), Ito et al. (2001), Ito et al. (2000), Newman et al. (2000), Ortiz et al. (1999), Uetz et al. (2000), Yoon and Carbon (1999), and Kang et al. (2001). Physical interactions between Mcm16-Ctf19, Mcm22-Ctf19, Mcm16-Ctf3, Mcm22-Ctf3, and Spc34-Mcm22 represent coimmunoprecipitation and two-hybrid data from V. Measday and P. Hieter (personal communication).
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fig6: Protein interactions establishing the connectivity between spindle microtubules and centromeric DNA. Physical interactions are indicated by lines between proteins. Shapes indicate distinct complexes within the kinetochore. Proteins that interact directly with centromeric DNA are shown in blue. Proteins whose loss of function results in genetic interactions with dam1-1 are shown in red. Protein interaction data are from Chen et al. (1998), Ghosh et al. (2001), Ito et al. (2001), Ito et al. (2000), Newman et al. (2000), Ortiz et al. (1999), Uetz et al. (2000), Yoon and Carbon (1999), and Kang et al. (2001). Physical interactions between Mcm16-Ctf19, Mcm22-Ctf19, Mcm16-Ctf3, Mcm22-Ctf3, and Spc34-Mcm22 represent coimmunoprecipitation and two-hybrid data from V. Measday and P. Hieter (personal communication).

Mentions: The identification of four additional subunits of the Dam1p complex allows for a more complete understanding of the roles that this complex plays in kinetochore function. Genome-wide two-hybrid screens identified multiple physical interactions between subunits of the Dam1p complex and other components of the kinetochore (Ito et al., 2000, 2001; Uetz et al., 2000). For example, this complex shows multiple interactions with components of the Ndc80 complex (Dam1p–Ndc80p, Spc19p–Ndc80p, and Spc19p–Nuf2p), suggesting that the functions of these two complexes may be closely associated. In addition, Dam1p interacts with Mcm16p (Ito et al., 2001), and Spc34p interacts by two-hybrid with Mcm22p (V. Measday and P. Hieter, personal communication). The interactions revealed by our genetic studies lend support to the biological relevance to the two-hybrid interactions with Mcm16p and Mcm22p and also point to an interaction with the Ctf19 complex. These data combined with recent work on other complexes within the kinetochore and the extensive protein–protein interactions identified by genome-wide two-hybrid studies, have helped to generate an appreciation of the large network of physical interactions that exist within the kinetochore. Fig. 6 represents the first attempt to model the physical interactions that may serve to connect microtubules and centromeric DNA in budding yeast. Although much work remains to be done, the elucidation of this network of interactions is an important first step toward understanding the organization and function of the outer kinetochore.


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)

Protein interactions establishing the connectivity between spindle microtubules and centromeric DNA. Physical interactions are indicated by lines between proteins. Shapes indicate distinct complexes within the kinetochore. Proteins that interact directly with centromeric DNA are shown in blue. Proteins whose loss of function results in genetic interactions with dam1-1 are shown in red. Protein interaction data are from Chen et al. (1998), Ghosh et al. (2001), Ito et al. (2001), Ito et al. (2000), Newman et al. (2000), Ortiz et al. (1999), Uetz et al. (2000), Yoon and Carbon (1999), and Kang et al. (2001). Physical interactions between Mcm16-Ctf19, Mcm22-Ctf19, Mcm16-Ctf3, Mcm22-Ctf3, and Spc34-Mcm22 represent coimmunoprecipitation and two-hybrid data from V. Measday and P. Hieter (personal communication).
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Related In: Results  -  Collection

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

fig6: Protein interactions establishing the connectivity between spindle microtubules and centromeric DNA. Physical interactions are indicated by lines between proteins. Shapes indicate distinct complexes within the kinetochore. Proteins that interact directly with centromeric DNA are shown in blue. Proteins whose loss of function results in genetic interactions with dam1-1 are shown in red. Protein interaction data are from Chen et al. (1998), Ghosh et al. (2001), Ito et al. (2001), Ito et al. (2000), Newman et al. (2000), Ortiz et al. (1999), Uetz et al. (2000), Yoon and Carbon (1999), and Kang et al. (2001). Physical interactions between Mcm16-Ctf19, Mcm22-Ctf19, Mcm16-Ctf3, Mcm22-Ctf3, and Spc34-Mcm22 represent coimmunoprecipitation and two-hybrid data from V. Measday and P. Hieter (personal communication).
Mentions: The identification of four additional subunits of the Dam1p complex allows for a more complete understanding of the roles that this complex plays in kinetochore function. Genome-wide two-hybrid screens identified multiple physical interactions between subunits of the Dam1p complex and other components of the kinetochore (Ito et al., 2000, 2001; Uetz et al., 2000). For example, this complex shows multiple interactions with components of the Ndc80 complex (Dam1p–Ndc80p, Spc19p–Ndc80p, and Spc19p–Nuf2p), suggesting that the functions of these two complexes may be closely associated. In addition, Dam1p interacts with Mcm16p (Ito et al., 2001), and Spc34p interacts by two-hybrid with Mcm22p (V. Measday and P. Hieter, personal communication). The interactions revealed by our genetic studies lend support to the biological relevance to the two-hybrid interactions with Mcm16p and Mcm22p and also point to an interaction with the Ctf19 complex. These data combined with recent work on other complexes within the kinetochore and the extensive protein–protein interactions identified by genome-wide two-hybrid studies, have helped to generate an appreciation of the large network of physical interactions that exist within the kinetochore. Fig. 6 represents the first attempt to model the physical interactions that may serve to connect microtubules and centromeric DNA in budding yeast. Although much work remains to be done, the elucidation of this network of interactions is an important first step toward understanding the organization and function of the outer kinetochore.

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