Hsp90 enables Ctf13p/Skp1p to nucleate the budding yeast kinetochore.
Binding of CBF3, a protein complex consisting of Ndc10p, Cep3p, Ctf13p, and Skp1p, to the centromere DNA nucleates kinetochore formation in budding yeast. Here, we investigate how the Ctf13p/Skp1p complex becomes competent to form the CBF3-centromere DNA complex. As revealed by mass spectrometry, Ctf13p and Skp1p carry two and four phosphate groups, respectively. Complete dephosphorylation of Ctf13p and Skp1p does not interfere with the formation of CBF3-centromere DNA complexes in vitro. Furthermore, deletion of corresponding phosphorylation sites results in viable cells. Thus, in contrast to the current view, phosphorylation of Ctf13p and Skp1p is not essential for the formation of CBF3-centromere DNA complexes. Instead, the formation of active Ctf13p/Skp1p requires Hsp90. Several lines of evidence support this conclusion: activation of heterologous Ctf13p/Skp1p by reticulocyte lysate is inhibited by geldanamycin and Hsp90 depletion. skp1 mutants exhibit growth defects on media containing geldanamycin. A skp1 mutation together with Hsp90 mutations exhibits synthetic lethality. An Hsp90 mutant contains decreased levels of active Ctf13p/Skp1p.
Stemmann
Olaf
O
Biochemie-Zentrum, Ruprecht-Karls Universität, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
Neidig
Anke
A
Köcher
Thomas
T
Wilm
Matthias
M
Lechner
Johannes
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000502:physiology*; D004268:DNA-Binding Proteins; D019281:Dimerization; D044783:F-Box Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D018841:HSP90 Heat-Shock Proteins / Q000502:physiology*; D018386:Kinetochores / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D044843:SKP Cullin F-Box Protein Ligases; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins
D001426:Bacterial Proteins; C081354:CTF13 protein, S cerevisiae; D004268:DNA-Binding Proteins; D044783:F-Box Proteins; D005656:Fungal Proteins; D018841:HSP90 Heat-Shock Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D044843:SKP Cullin F-Box Protein Ligases; C120507:SKP1 protein, S cerevisiae