Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization

Abstract

The location of proteins within eukaryotic cells is often critical for their function and relocation of proteins forms the mainstay of regulatory pathways. To assess the importance of protein location to cellular homeostasis, we have developed a methodology to systematically create binary physical interactions between a query protein and most other members of the proteome. This method allows us to rapidly assess which of the thousands of possible protein interactions modify a phenotype. As proof of principle we studied the kinetochore, a multiprotein assembly that links centromeres to the microtubules of the spindle during cell division. In budding yeast, the kinetochores from the 16 chromosomes cluster together to a single location within the nucleus. The many proteins that make up the kinetochore are regulated through ubiquitylation and phosphorylation. By systematically associating members of the proteome to the kinetochore, we determine which fusions affect its normal function. We identify a number of candidate kinetochore regulators, including the phosphatase Cdc14. We examine where within the kinetochore Cdc14 can act and show that the effect is limited to regions that correlate with known phosphorylation sites, demonstrating the importance of serine phospho-regulation for normal kinetochore homeostasis.T he relocation of proteins between cellular compartments underlies many regulatory pathways. For example, protein relocation underpins most cell-signaling pathways (1) and the establishment of cell polarity and asymmetric cell division both require highly specialized relocation of proteins within the cell (2). Furthermore, the aberrant localization of proteins underlies the pathology of a number of diseases (3). However, our understanding of the effect of relocalizing members of the proteome is limited to specific studies typically concerning individual proteins, and only a select few studies have globally monitored protein relocation (4, 5).One highly localized structure within the cell is the kinetochore, which in budding yeast forms a single megadalton complex (6-8). The kinetochore attaches chromosomes to the spindle microtubules to drive accurate chromosome segregation. The kinetochore consists of at least 60 unique gene products present in multiple copies that stretch from the specialized H3 histone subunit (Cse4, CENP-A in mammals) to the microtubule binding proteins of the Dam1 complex (9). Kinetochores normally assemble hierarchically from the centromere-bound proteins (10). Once assembled, the structural homeostasis of the kinetochore is regulated by proteins that are recruited to kinetochores. For example, the histone subunit Cse4 is tightly regulated by the E3 ubiquitin ligase Psh1, which is localized at centromeres. Psh1 prevents excessive Cse4 centromere loading via ubiquitylation-dependent degradation of Cse4 (and at noncentromeric regions) (11). Cse4 is also phosphorylated by Ipl1, likely to destabilize aberrant microtubule interactions and ensure correct sister chromosome biorientation (12...

Protein Kinase C and the stress response pathways are required for kinetochore homeostasis

“…We confirmed that Pkc1-GBP was co-localizing with endogenously GFP-tagged kinetochore using fluorescence imaging. We found that the GFP-GBP binding is sufficient to cause considerable relocalization of Pkc1 to the kinetochore in all cases (Fig 5A), consistent with previous observations (Berry et al, 2016; Olafsson & Thorpe, 2015). We found that a number of haploid strains, which contain only a single endogenously encoded copy of the GFP tagged protein, are restricted, for growth when expressing Pck1-GBP.…”

“…The fluoresecence intensity within a ~600 nm square box in the focal plane of each foci is measured and a local background signal for each focus is subtracted. We found that the deviations in fluorescence intensity of control strains across each 48 pad imaging plate and consequently we used a smoothing algorithm to normalize values across each plate of the screen (Olafsson & Thorpe, 2015).…”

“…We used the established method for synthetic physical interactions (Olafsson & Thorpe, 2015). In brief, The GBP / PKC1-GBP alleles were expressed from a single copy CEN plasmid under the control of a CUP1 promoter, all strains were grown at 30°C.…”

“…Whole cell extracts where prepared by using trichloracetic acid (TCA) as previously described (Olafsson & Thorpe, 2015). Protein extracts were separated on acrylamide gels (7.5% for Pkc1 and 10% for Dad3, Dad4 and Mtw1).…”

“…However, we wanted to ask whether forced recruitment of Pkc1 to the kinetochore would lead to a mitotic phenotype. To test this, we made use of the Synthetic Physical Interaction (SPI) system to separately associate Pkc1 with kinetochore and associated proteins (Olafsson & Thorpe, 2015). The SPI system uses an antibody fragment that binds to GFP, the GFP-binding protein (GBP), which is fused to Pkc1, to bind to GFP-tagged proteins in the cell.…”