Sgt1 dimerization is required for yeast kinetochore assembly.
The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study, we show that Sgt1 forms homodimers by performing in vitro and in vivo immunoprecipitation and analytical ultracentrifugation analyses. Analyses of the dimerization of Sgt1 deletion proteins showed that the Skp1-binding domain (amino acids 1-211) contains the Sgt1 homodimerization domain. Also, the Sgt1 mutant proteins that were unable to dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is important for Sgt1-Skp1 binding. Restoring dimerization activity of a dimerization-deficient sgt1 mutant (sgt1-L31P) by using the CENP-B (centromere protein-B) dimerization domain suppressed the temperature sensitivity, the benomyl sensitivity, and the chromosome missegregation phenotype of sgt1-L31P. These results strongly suggest that Sgt1 dimerization is required for kinetochore assembly.
Bansal
Parmil K
PK
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-3678, USA.
Nourse
Amanda
A
Abdulle
Rashid
R
Kitagawa
Katsumi
K
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D002455:Cell Division / Q000502:physiology*; D015825:Chromosomes, Fungal / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018386:Kinetochores / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
D048868:Adaptor Proteins, Signal Transducing; C081354:CTF13 protein, S cerevisiae; C074510:HSP82 protein, S cerevisiae; D018841:HSP90 Heat-Shock Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D012097:Repressor Proteins; C470726:SGT1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins