Kinetochore assembly

The kinetochore is a protein-DNA machine that links chromosomes to the mitotic spindle. The large number of proteins required for kinetochore function suggest that it behaves as a network, where each complex effects the integrity and function of the rest. The cartoon below shows the centromeric heterochromatin at the bottom and the attachment complexes that link this part of the chromosome to protein complexes that mediate microtubule attachment. 


adapted from Ciferri et al., FEBS Letters, 2007

Multi-protein network: The Mis12 complex is highly conserved in eukaryotes and acts as a "keystone" complex, interacting with other multi-protein complexes that are proximal to both DNA and microtubules. A variety of genetic and function studies suggest that the Mis12 complex stabilizes the kinetochore network. Importantly, we have found that the Hsp90-Sgt1 chaperone complex is required to efficiently assembly the Mis12 complex and thus kinetochores (Davies and Kaplan, JCB 2010). 

Hsp90-Sgt1 assembly pathway: This same pathway is conserved in budding yeast where Hsp90-Sgt1 is important for the assembly of the CBF3 complex, another "keystone" like complex that is required for stabilizing the entire kinetochore network (Rodrigo-Brenni et al., MBC 2004). 

Our data from human cells and from budding yeast is consistent with the following model for multi-complex assembly. Hsp90-Sgt1 interacts with an assembly intermediate. In this case illustrated below, this is represented by a partial assembly of a multi-protein complex (light green and yellow subunits). Hsp90-Sgt1 holds this assembly intermediate in a stable conformation to allow the specific high affinity interactions with other subunits of the complex (conformation #1). Assembly releases Hsp90 in this model and allows the accurate assembly of one multi-protein complex (conformation #2), in the case of kinetochores either CBF3 in yeast or Mis12 in humans. 


Quality control of protein network assembly: Evidence from both budding yeast and human cells demonstrate that assembly mediated by Hsp90-Sgt1 is linked to ubiquitin-mediated degradation pathway. For kinetochores, this pathway requires the SCF ubuiqitin ligase. The turnover of kinetochore subunits by degradation is uncovered by inhibiting the Hsp90-Sgt1 assembly pathway. We propose that unassembled complexes are targeted for ubiquitin modification and turnover when they fail to find their appropriate binding partner. Thus, partially assembled or mis-assembled complexes do not become incorprated into the network. 

 

In the micographs below, the normal balance of assembly and turnover pathways results in functional kinetochores formed in a timely manner (micrograph on left). When the assembly pathway is blocked, kinetochores fail to form in a timely manner (middle micrograph). When both assemlby and turnover are blocked kinetochore proteins are incorporated into kinetochores quickly but there are less robust microtubule binding sites (micrograph on right), suggesting the kinetochore network has been compromised. 


Future directions: 

1. We want to know how Hsp90-Sgt1 stabilizes assembly intermediates by reconstituting assembly steps in vitro with purified recombinant proteins. 

2. We want to understand the mechanisms that link assembly to SCF-mediated ubiquitination. 

3. We want to understand how cell "stress" - variations from homeostasis - affect the rules of Hsp90-mediated protein complex assembly, focusing on the implications for stress-induced genomic instability (Kaplan and Li, Trends in Cell Biology, 2012). 




kbkaplan@ucdavis.edu © K.B. Kaplan 2012