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Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)-related protein Sli15 during chromosome segregation.

Kang J, Cheeseman IM, Kallstrom G, Velmurugan S, Barnes G, Chan CS - J. Cell Biol. (2001)

Bottom Line: Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants.Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo.Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome-spindle interactions or in the movement of kinetochores along microtubules.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, The University of Texas, Austin, TX 78712, USA.

ABSTRACT
We have shown previously that Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. We show here that Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1-Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome-spindle interactions or in the movement of kinetochores along microtubules.

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GST–Ipl1 and GST–Sli15-M cosediment with microtubules. Different concentrations of taxol-stabilized microtubules were incubated with GST (A), GST–Ipl1 (B), or GST–Sli15-M (C). The reactions were then centrifuged at high speed to generate the supernatant (S) and pellet (P) fractions. The presence of GST or GST fusion protein in either fraction was determined by immunoblotting with anti-GST antibodies. The percentage of GST–Ipl1 or GST–Sli15-M that was bound to and copelleted with the microtubules in each reaction was quantitated by densitometry of the immunoblots. The binding curves display the average and range of values obtained from duplicate experiments.
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fig4: GST–Ipl1 and GST–Sli15-M cosediment with microtubules. Different concentrations of taxol-stabilized microtubules were incubated with GST (A), GST–Ipl1 (B), or GST–Sli15-M (C). The reactions were then centrifuged at high speed to generate the supernatant (S) and pellet (P) fractions. The presence of GST or GST fusion protein in either fraction was determined by immunoblotting with anti-GST antibodies. The percentage of GST–Ipl1 or GST–Sli15-M that was bound to and copelleted with the microtubules in each reaction was quantitated by densitometry of the immunoblots. The binding curves display the average and range of values obtained from duplicate experiments.

Mentions: Because Ipl1 and Sli15 both localize along the length of the mitotic spindle (Biggins et al., 1999; Kim et al., 1999), we tested whether either of these proteins can bind to microtubules directly by examining the ability of GST and GST fusion proteins (that were purified from E. coli) to cosediment with taxol-stabilized microtubules in vitro. GST did not sediment with microtubules (Fig. 4 A). In contrast, whereas only a small fraction of GST–Ipl1 sedimented in the absence of microtubules, the bulk of GST–Ipl1 did cosediment with microtubules in a microtubule concentration–dependent manner (Fig. 4 B). The estimated dissociation constant of the GST–Ipl1–microtubule interaction is ∼0.5 μM, which represents a slightly lower affinity than that between tau and microtubules (Goode and Feinstein, 1994).


Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)-related protein Sli15 during chromosome segregation.

Kang J, Cheeseman IM, Kallstrom G, Velmurugan S, Barnes G, Chan CS - J. Cell Biol. (2001)

GST–Ipl1 and GST–Sli15-M cosediment with microtubules. Different concentrations of taxol-stabilized microtubules were incubated with GST (A), GST–Ipl1 (B), or GST–Sli15-M (C). The reactions were then centrifuged at high speed to generate the supernatant (S) and pellet (P) fractions. The presence of GST or GST fusion protein in either fraction was determined by immunoblotting with anti-GST antibodies. The percentage of GST–Ipl1 or GST–Sli15-M that was bound to and copelleted with the microtubules in each reaction was quantitated by densitometry of the immunoblots. The binding curves display the average and range of values obtained from duplicate experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2150868&req=5

fig4: GST–Ipl1 and GST–Sli15-M cosediment with microtubules. Different concentrations of taxol-stabilized microtubules were incubated with GST (A), GST–Ipl1 (B), or GST–Sli15-M (C). The reactions were then centrifuged at high speed to generate the supernatant (S) and pellet (P) fractions. The presence of GST or GST fusion protein in either fraction was determined by immunoblotting with anti-GST antibodies. The percentage of GST–Ipl1 or GST–Sli15-M that was bound to and copelleted with the microtubules in each reaction was quantitated by densitometry of the immunoblots. The binding curves display the average and range of values obtained from duplicate experiments.
Mentions: Because Ipl1 and Sli15 both localize along the length of the mitotic spindle (Biggins et al., 1999; Kim et al., 1999), we tested whether either of these proteins can bind to microtubules directly by examining the ability of GST and GST fusion proteins (that were purified from E. coli) to cosediment with taxol-stabilized microtubules in vitro. GST did not sediment with microtubules (Fig. 4 A). In contrast, whereas only a small fraction of GST–Ipl1 sedimented in the absence of microtubules, the bulk of GST–Ipl1 did cosediment with microtubules in a microtubule concentration–dependent manner (Fig. 4 B). The estimated dissociation constant of the GST–Ipl1–microtubule interaction is ∼0.5 μM, which represents a slightly lower affinity than that between tau and microtubules (Goode and Feinstein, 1994).

Bottom Line: Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants.Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo.Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome-spindle interactions or in the movement of kinetochores along microtubules.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, The University of Texas, Austin, TX 78712, USA.

ABSTRACT
We have shown previously that Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. We show here that Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1-Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome-spindle interactions or in the movement of kinetochores along microtubules.

Show MeSH