L-1 phosphorylation by PLK-1 on interaction with BUB-1 and BUB-3 by incubating beads coated with GST-tagged KNL-11505 in a reticulocyte lysate expressing BUB-11494 and BUB-3. Phosphorylation by PLK-1 increased association of BUB-1 and BUB-3 with KNL-11505 by 2.4 and 3.8 fold respectively. Thus, phosphorylation of KNL-1 by PLK-1 promotes interaction of the KNL-1 Nterminus with BUB-1 and BUB-3. Cell Rep. Author manuscript; available in PMC 2016 July 07. Espeut et al. Page 4 To assess the contribution of the MELT repeats to the phosphorylation PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850275 of the KNL-1 Nterminus, we compared PLK-1 kinase activity on WT KNL-11505 to a mutant with the 11 MELT repeats mutated to AEAA. Mutation of the MELT repeats reduced KNL-11505 phosphorylation to ~60 % of WT KNL-11505 indicating that additional sites are targeted by PLK-1. To identify these other sites, we analysed phosphorylation of recombinant fragments followed by targeted amino acid mutations. Using this approach, we identified 8 sites phosphorylated by PLK-1, 
whose mutation to alanine decreased phosphorylation of KNL-11505 by ~50%. Combining mutation of the MELT repeats and of the 8 additional sites, additively reduced PLK-1 phosphorylation to ~20% of control. Thus, biochemical analysis defined a set of residues whose mutation should enable testing the functional significance of PLK-1 phosphorylation of KNL-1 in vivo. KNL-1 Mutants Compromised for PLK-1 Phosphorylation Retain Functional Properties Associated with the KNL-1 N-terminus As the mutations introduced to reduce PLK-1 phosphorylation alter a significant number of residues in KNL-1, we were concerned about interpreting such mutants in vivo. The KNL-1 N-terminus has a PP1 docking site, a microtubule-binding activity, and behaves as an oligomer on gel filtration. We therefore tested all three properties for the MELT/A, 8A and MELT/A+8A mutations and found that none of them was affected by mutations in PLK-1 phosphorylation sites. Thus, any effect of these mutations in vivo is unlikely to be due to a non-specific disruption of the N-terminal half of KNL-1. A KNL-1 Mutant Compromised for PLK-1 Phosphorylation Significantly Reduces BUB-1 Kinetochore Recruitment We next generated strains expressing single copy RNAi-resistant versions of MELT/A, 8A and MELT/A+8A mutant forms of KNL-1 in vivo. These transgenes were based on a prior RNAi-resistant knl-1::mCh transgene that was functionally validated. The three KNL-1 mutants generated–MELT/A, 8A and MELT/A+8A–all localized to kinetochores at levels similar to WT KNL-1. To monitor BUB-1 kinetochore localization in these mutants, we introduced a bub-1::gfp transgene into the different knl-1::mCh transgene containing strains, depleted endogenous KNL-1, and measured BUB-1::GFP levels relative to KNL-1::mCherry on kinetochores of aligned chromosomes. This analysis revealed that the 8A and MELT/A mutants recruited less BUB-1 at kinetochores compared to WT KNL-1. Neuromedin N Notably, in the MELT/A+8A mutant, significantly less BUB-1 was recruited to kinetochores, compared to MELT/A or 8A alone. Thus, mutations that compromise PLK-1 phosphorylation of the KNL-1 Nterminus in vitro significantly perturb BUB-1 kinetochore recruitment in vivo, with the MELT/A+8A mutant nearly abolishing BUB-1 localization. To compare the ability of WT and mutant KNL-1 to support chromosome segregation, we crossed in GFP fusions with histone H2b and -tubulin and depleted endogenous KNL-1 Author Manuscript Author Manuscript Author Manuscri