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Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

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Sgt1 dimerization is required for yeast kinetochore assembly.

Parmil K Bansal¹, Amanda Nourse, Rashid Abdulle

¹Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-3678, USA.

The Journal of Biological Chemistry

|December 17, 2008

Summary

Sgt1 protein dimerization is crucial for kinetochore assembly and accurate chromosome segregation during cell division. Restoring this dimerization corrects defects in budding yeast.

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Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

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Published on: May 3, 2015

Area of Science:

Cell Biology
Molecular Biology
Genetics

Background:

The kinetochore is vital for accurate chromosome transmission during cell division.
In budding yeast, Sgt1 and Hsp90 facilitate the assembly of the core kinetochore complex CBF3.
Sgt1 and Hsp90 activate CBF3 components Skp1 and Ctf13.

Purpose of the Study:

To investigate the role of Sgt1 dimerization in kinetochore assembly.
To determine the specific domain responsible for Sgt1 homodimerization.
To assess the functional significance of Sgt1 dimerization in vivo.

Main Methods:

In vitro and in vivo immunoprecipitation assays.
Analytical ultracentrifugation.
Analysis of Sgt1 deletion and mutant proteins.
Functional complementation assays using CENP-B dimerization domain.

Main Results:

Sgt1 protein forms homodimers.
The Skp1-binding domain (amino acids 1-211) is essential for Sgt1 homodimerization.
Sgt1 dimerization is required for Skp1 binding.
Restoring dimerization in a mutant sgt1 protein corrected temperature sensitivity, benomyl sensitivity, and chromosome missegregation.

Conclusions:

Sgt1 dimerization is essential for its interaction with Skp1.
Sgt1 homodimerization is a prerequisite for proper kinetochore assembly.
Sgt1 dimerization plays a critical role in maintaining genomic stability during cell division.