Molecular Cloning and Mutagenesis of Ubiquitin and α-Synuclein

Human ubiquitin was polymerase chain reaction (PCR)–amplified from pcDNA3-HA-ubiquitin (gift from E. Yeh, Addgene plasmid #18712) (50 (link)) and cloned with a VN tag into pcDNA3. Subsequently, α-synuclein–VC and VN-ubiquitin were cloned into a single backbone with pIRES2 to drive simultaneous expression of both proteins with the same promoter, using the NEBuilder HiFi DNA assembly [New England Biolabs (NEB)] kit. Rab5^Q79L was generated by site-directed mutagenesis of mRFP-Rab5 (gift from A. Helenius, Addgene plasmid #4437) (51 (link)), using the QuikChange II site-directed mutagenesis kit (Agilent). mRFP-NBR1 was generated by replacing Rab5 cDNA in the mRFP-Rab5 plasmid with NBR1 cDNA amplified from pMXs-puro-mCherry-NBR1 (gift from J. Debnath, Addgene plasmid #74242) (52 (link)) using the NEBuilder HiFi DNA assembly (NEB) kit. mRFP-NBR1^ΔUBD was generated by adapting mRFP-NBR1 using the QuikChange site-directed mutagenesis kit. WT α-synuclein–pHluorin was cloned using pCMV-lyso-pHluorin (gift from C. Rosenmund, Addgene plasmid #70113) (53 (link)), and 3KR was generated using the QuikChange II site-directed mutagenesis kit (Agilent). PCR-based site-directed mutagenesis was also performed to introduce mutations in VN-ubiquitin (K48R, K63R, K48R/K63R, and ΔGG) and α-synuclein–VC (3KR, 4KR, and S129A). All newly constructed plasmids were validated by sequencing (Eurofins).