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The role of Saccharomyces cerevisiae Bir1p within the Ipl1p protein kinase complex required for execution of mitosis

Reference	BB/C007077/1
Principal Investigator / Supervisor	Professor Michael Stark
Co-Investigators / Co-Supervisors
Institution	University of Dundee
Department	School of Life Sciences
Funding type	Research
Value (£)	191,582
Status	Completed
Type	Research Grant
Start date	18/07/2005
End date	17/07/2008
Duration	36 months

Abstract

Ipl1p is a protein kinase that is critically involved in regulation of the yeast cell division cycle and recent work has shown that it is required to ensure that all chromosomes become properly attached to the mitotic spindle prior to division. The majority of replicated yeast chromosomes start out with both sister kinetochores attached to microtubules originating from the old spindle pole, and such mono-oriented, syntelic attachments need to be corrected to yield a bi-oriented configuration in which sister kinetochores are bound by microtubules emanating from opposite poles. IpI1p catalyses such conversion, apparently by phosphorylating critical substrates at the kinetochore such as Dam1p. Ipl1p is found in a complex with two other proteins, SIi15p and Bir1p, but while SIi15p is well established as an activator of IpI1p kinase and as playing a role in the localisation of the kinase complex, in contrast little is known about the role of Bir1p. This proposal will address the function of Bir1p within the IpI1p- SIi15p-Bir1p complex by adopting several complementary approaches as summarised below: (1) Testing the dependence of IpI1p function on its association with Bir1p. By making mutants deficient in Bir1p function we will examine which functions of the complex are dependent on Bir1p. In particular, we wish to demonstrate whether Bir1p association is critically required for promoting chromosome bi-orientation and whether Bir1p plays a role in regulating the subcellular localisation of the complex, which binds initially to kinetochores and then relocalises to the spindle as cells complete mitosis. Bi-orientation will be monitored by examining the behaviour of sister centromere sequences that have been tagged with GFP and localisation of the complex will utilise GFP-tagged IpI1p and/or SIi15p. (2) Interactions within the IpI1p-SIi15p-Bir1p complex. We will examine the interactions between the three components by using a combination of radiolabelled, in vitro translatedand GST-tagged proteins to determine whether Bir1p binds to SIi15p, IpI1p or both and to map the interactions more precisely by making subclones expressing truncated proteins to determine which domains are involved. (3) The role of Bir1p in the kinase activity of the complex. We will test the effect of Bir1p on the intrinsic activity of the Ipl1p- SIi15p complex using recombinant proteins expressed in bacteria and/or complexes assembled from TAP-tagged components expressed and purified from yeast. (4) Phosphorylation of Bir1p. We will map the location of phosphate groups on Bir1p isolated from yeast cells arrested in mitosis by inactivation of Anaphase Promotion Complex (Cdc20p depletion) and then mutate the sites to study the significance of phosphorylation for Bir1p and kinase complex function.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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