Nebuilder hifi assembly

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.

Bcl2 overexpression vector was generated by cloning Bcl2 cDNA (Transomic Technologies, Cat. TCM1304) into the pMSCV-loxp-dsRed-loxP-eGFP-puro-WPRE vector (Addgene #32702) using the EcoRI and NsiI restriction sites. Ankle2 cDNA (Transomic Technologies, Cat. TCM1004) was cloned into the MSCV-IRES-Thy1.1 vector using NEBuilder Hifi Assembly (New England Biolabs). All vectors were propagated in Stbl3 cells (ThermoFisher Scientific).

The mRNA injection assay and the modified pRNA destination vector with the C-terminal mKate2 tag used here have been described previously (Novak et al., 2014 (link); Novak et al., 2016 (link)). Spd-2-ALL and Spd-2-CONS cDNA were introduced into the vector via Gateway cloning. Two point mutations were introduced into WT Spd-2-mKate2 using QuikChange mutagenesis (Agilent) to generate Spd-2-AA-mKate2. Spd-2-NT-mKate2 and Spd-2-CT-mKate2 partial mutants were derived from PCR-amplified fragments of WT Spd-2-mKate2 and Spd-2-ALL-mKate2 via NEBuilder HiFi assembly (New England Biolabs). The last potential binding site mutated in the N-terminus group was S538-S540, and the first potential binding site mutated in the C-terminus group was S581-S582, so that each group would include 17 potential sites. In vitro RNA synthesis was performed using a T3 mMESSAGE mMACHINE kit (Thermo Fisher) and RNA was purified using an RNeasy MinElute kit (Qiagen). All RNA constructs were injected at a concentration of 2 mg/mL.

The basal hsp68 promoter of PCR-linearized p130der was replaced with the TC006550 promoter amplified from blaAmp-Tc6550Pro-GFPZeo-Luciferase-HSP-Orange-pIZT (kindly provided by Dr. Yoonseong Park) [13 (link)] with NEBuilder HiFi assembly (NEB). The resulting pTC006550-GAL4Δ-SV40 polyA coding sequence was then amplified and inserted into PCR-linearized pBac[3xP3-EGFP] with NEBuilder HiFi assembly. (DGRC # 1427)

TurboID was a kind gift of A. Ting (Addgene #107171)^{29 (link)}. PMLIVa^WT and PMLIVa^3MAS were previously described^{21 (link)}. SUMO1, SUMO2, Ub, RANGAP1 and UBC9 ORFs were amplified from U2OS cell cDNA by high-fidelity PCR (Platinum SuperFi DNA Polymerase; Invitrogen). A GSQ linker (GGGSSGGGQISYASRG) was placed between the C-terminal part of TurboID and the UbLs as well as between the N-terminal part and the substrates. All constructs were generated by standard cloning or by Gibson Assembly (NEBuilder HiFi Assembly, NEB) using XL10-Gold bacteria (Agilent). Depending on the construction, plasmid backbones derived from EYFP-N1 (Clontech/Takara), Lenti-Cas9-blast (a kind gift of F. Zhang; Addgene #52962) or TRIPZ (Open Biosystems/Horizon) were used. After assembly, all vectors were validated by sequencing. Additional details for constructs are described in Supplementary Table 1. Oligonucleotides sequences are shown in Supplementary Table 2. The sequences of representative constructs are in the Source Data file. Cloning details about other constructs are available upon request.

Plasmids used in this study are listed in Table S2. Plasmids were constructed using NEBuilder® HiFi Assembly (New England Biolabs, Ipswich, MA, USA). Phusion® High‐Fidelity Polymerase (New England Biolabs, Ipswich, MA, USA) or PrimeSTAR® Max High‐Fidelity Polymerase (Takara Bio, Mountain View, CA, USA) was used to amplify DNA for cloning and genome integration. Quick‐DNA Miniprep and Zyppy™ Plasmid Miniprep Kits were used to purify genomic and plasmid DNA (Zymo Research, Irvine, CA, USA). DNA Clean and Concentrator, and Zymoclean™ Gel DNA Recovery Kits were used to purify PCR fragments (Zymo Research, Irvine, CA, USA). Restriction enzymes were purchased from New England Biolabs. Plasmids were confirmed by restriction digest and sequencing (ACGT, Inc., Wheeling, IL, USA).
To construct 44_ediss from 445_ediss gifted by Stefan Pflügl [35 ], primers were used to amplify a linear fragment containing the plasmid backbone, budA, and budB, then subsequently circularized to create the budB‐budA operon. The new plasmid 44_ediss was confirmed by restriction digest and sequencing.