Sgt1 Associates with Hsp90: an Initial Step of Assembly of the Core Kinetochore Complex

Abstract

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for highfidelity chromosome transmission during cell division. In budding yeast, Sgt1, together with Skp1, is required for assembly of the core kinetochore complex (CBF3) via Ctf13 activation. Formation of the active Ctf13-Skp1 complex also requires Hsp90, a molecular chaperone. We have found that Sgt1 interacts with Hsp90 in yeast. We also have determined that Skp1 and Hsc82 (a yeast Hsp90 protein) bind to the N-terminal region of Sgt1 that contains tetratricopeptide repeat motifs. Results of sequence and phenotypic analyses of sgt1 mutants strongly suggest that the N-terminal region containing the Hsc82-binding and Skp1-binding domains of Sgt1 is important for the kinetochore function of Sgt1. We found that Hsp90's binding to Sgt1 stimulates the binding of Sgt1 to Skp1 and that Sgt1 and Hsp90 stimulate the binding of Skp1 to Ctf13, the F-box core kinetochore protein. Our results strongly suggest that Sgt1 and Hsp90 function in assembling CBF3 by activating Skp1 and Ctf13.The centromere is essential for chromosome inheritance and requires DNA sequence elements and structural and regulatory proteins (the structural protein complex is called the kinetochore) for its activity and coordination within the cell cycle. In budding yeast (Saccharomyces cerevisiae), the kinetochore consists of more than 30 different proteins, and several of these proteins are conserved in mammals (21). The budding yeast centromere DNA is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (8, 13). CDEIII (25 bp) is essential for centromere function (12) and was the site shown to be bound by CBF3, a protein complex that contains the major proteins p110 (encoded by NDC10/ CTF14/CBF2), p64 (encoded by CEP3/CBF3b), p58 (encoded by CTF13) (4,5,11,17,23,24,37,39), and Skp1 (4, 37). All four are essential for viability, and mutations in any one abolish the CDEIII-binding activity of CBF3 (18,36). No known kinetochore protein, except for CDEI-binding Cbf1, can localize to kinetochores when the CBF3 complex is disrupted. Therefore, the CBF3 complex is the fundamental structure of the kinetochore, and the mechanism of CBF3 assembly is of great interest.SGT1 was isolated as a dosage suppressor of skp1-4, a kinetochore-defective mutant (22). By activating Ctf13, Sgt1 and Skp1 are required for assembly of the CBF3 complex (22). However, the mechanism of activation is not well characterized. Stemmann et al. showed that formation of the active Ctf13-Skp1 complex requires Hsp90, a molecular chaperone (38). Sgt1 has highly conserved tetratricopeptide repeat (TPR) (22) and CS (CHORD protein and Sgt1-specific) motifs-in other proteins these motifs are required for interaction with Hsp90 (2, 6). Here we report that molecular chaperones from the Hsp90 and Hsp70 families form a complex with Sgt1 in yeast. Our results strongly suggest that Hsp90's interaction with Sgt1 is required for kinetochore assembly in yeast. On the basis of our findi...

Structural and Functional Analysis of SGT1 Reveals That Its Interaction with HSP90 Is Required for the Accumulation of Rx, an R Protein Involved in Plant Immunity

“…First, it is clear that SGT1 is able to interact directly with HSP90 even in the absence of RAR1 ( Figure 9A), as shown by the yeast two-hybrid analysis and in vitro pull-down assays. Consistent with this, SGT1 interacts with HSP90 in yeast, which does not contain any RAR1 homologs (Bansal et al, 2004). The fact that the HSP90 interaction surface of the CS domain, but not that of RAR1, is highly conserved in eukaryotes also supports this notion.…”

“…Like HSP90, both SGT1 and RAR1 are required for the stabilization of many R proteins (Tornero et al, 2002;Bieri et al, 2004;Azevedo et al, 2006). Similarly, both HSP90 and SGT1 are required for the stabilization and assembly of the yeast CBF3 (for centromere binding factor3) and the human kinetochore complexes (Bansal et al, 2004;Lingelbach and Kaplan, 2004;Steensgaard et al, 2004;Niikura et al, 2006). As yeast does not contain any RAR1 homolog, CBF3 complex assembly is a RAR1-independent process.…”

“…Whether CHP1, a RAR1 homolog in human, is required for the kinetochore complex assembly is not known, although the Aspergillus homolog CHPA seems not to be involved in this process (Sadanandom et al, 2004). Interestingly, SGT1 is also involved in degradative processes, as it is required for the function of several SCF (for SKP1/CULLIN1/F-box protein) complexes in Saccharomyces cerevisiae and plants (Kitagawa et al, 1999;Gray et al, 2003;Bansal et al, 2004). In these organisms, SGT1 interacts with SCF subunit SKP1, indicating its role in the degradation of a potentially large number of proteins.…”

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

“…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). Hsp90 and Sgt1 are not core kinetochore components and only transiently associate with Ctf13.…”

The Hsp90 chaperone machinery: from structure to drug development

“…Sgt1 functions as an adaptor linking various target proteins to Hsp90 (27). The TPR domain of Sgt1 interacts with Skp1 in the context of the CBF3 kinetochore complex or SCF E3 ubiquitin ligase (31,32). In addition, through its SGS domain, Sgt1 can interact with many LRR (leucine rich repeat) -containing proteins, such as yeast adenylate cyclase Cdc35 (33), plant R proteins (27,34) and mammalian NLR (Nod-like receptor) proteins including Nod1 and Nalp3 (35)(36)(37).…”