Identifying Polo-binding Sites in Centrosomal Proteins

Potential Polo-binding sites in the amino acid sequence of the candidate centrosomal proteins were identified by searching for the consensus Polo-binding motif S-S/T. Site conservation was assessed using FlyBase BLAST (selecting the genus Drosophila) and Jalview (Waterhouse et al., 2009 (link)) for protein alignment. The mutant constructs were designed in silico and synthesised externally by GENEWIZ Co. Ltd. (Suzhou, China); the WT cDNAs were obtained from the Drosophila Genomics Resource Centre, USA. All cDNAs were cloned into a pDONR-Zeo vector and then introduced via Gateway cloning (Thermo Fisher Scientific; 11789100 and 11791100) in pRNA-mKate2CT (Novak et al., 2016 (link)) or Ubq-GFPCT and Ubq-mCherryCT (Basto et al., 2008 (link)) destination vectors, as indicated. NEBuilder HiFi assembly (NEB; E2621S) was used to produce pRNA-mKate2 plasmids encoding Ana1 ‘partial mutants’ and to introduce fragments encoding WT or mutant Ana1 amino acids 1431–1729 into a pETM44 (EMBL) vector encoding an N-terminal His6–MBP tag.