Hsp90 enables Ctf13p/Skp1p to nucleate the budding yeast kinetochore

Abstract

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 Ctf13p͞ 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 Ctf13p͞Skp1p requires Hsp90. Several lines of evidence support this conclusion: activation of heterologous Ctf13p͞Skp1p 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 Ctf13p͞Skp1p.

Sgt1p and Skp1p Modulate the Assembly and Turnover of CBF3 Complexes Required for Proper Kinetochore Function

“…Our results indicate that Skp1p and Sgt1p form important contacts with each other and with the F-box protein Ctf13p that allow Ctf13p to associate with Ndc10p and ultimately bind CEN DNA. Two models have been proposed to explain the "activation" of Ctf13p: 1) posttranslational modification in the form of phosphorylation , and 2) the recruitment of HSP90 chaperones (Stemmann et al, 2002). Our efforts to distinguish between these two possibilities suggest that Ctf13p activation may be controlled by multiple pathways.…”

“…Biochemical and genetic studies suggest that Skp1p and Sgt1p modify Ctf13p, possibly through protein phosphorylation or the action of HSP90 type chaperones Stemmann et al, 2002). However, the precise biochemical changes required for Ctf13p activation and how activation contributes to CBF3 function remain unclear.…”

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…The activation process requires binding to Skp1, a protein that is also a component of the SCF ubiquitin ligase complex (Bai et al 1996;Connelly and Hieter 1996;Kaplan et al 1997). Although the activation process was initially thought to require Ctf13 phosphorylation by a Skp1-interacting kinase , later work showed that phosphorylation is not required for CBF3 assembly on centromeres (Stemmann et al 2002). Instead, an Hsp90-Sgt1 co-chaperone complex binds to Skp1, which enhances Skp1 binding to Ctf13 (Stemmann et al 2002;Bansal et al 2004;Rodrigo-Brenni et al 2004).…”

Molecular Chaperone Hsp90 Associates with Resistance Protein N and Its Signaling Proteins SGT1 and Rar1 to Modulate an Innate Immune Response in Plants

“…SGT1 proteins also contain a tetratricopeptide repeat (TPR) domain, which is an Hsp90-binding domain in co-chaperones (14). SGT1 and Rar1 might associate with Hsp90 to play a chaperone-like function in the assembly or the conformational regulation of diverse multiprotein complexes (15). Interestingly, in a yeast two-hybrid screen with Nicotiana benthamiana Rar1 (NbRar1) as bait, NbSGT1 and NbHsp90 were identified as interacting proteins (9).…”