Appropriate restriction enzymes

Full-length PSAT genes of S. cerevisiae (ScPSAT; NCBI ID: AJT97493.1) were acquired. The yeast PSAT-encoding genes were amplified by PCR using genomic DNA obtained from the Korean Collection for Type Cultures as a template, Pfu-X DNA polymerase (SolGent, Daejeon, Republic of Korea), and oligonucleotide primers (Cosmo Genetech Inc., Seoul, Republic of Korea). The amplified products were digested with appropriate restriction enzymes (Enzynomics, Daejeon, Republic of Korea) at 37 °C for 3 h. The digested products were ligated with the pET28a vector using T4 ligase (M0202S; Roche, Basel, Switzerland) at 18 °C overnight and subsequently transformed into E. coli strain DH5α. The transformants were confirmed by colony PCR and DNA sequencing.

We used the pCMV-3Tag-6 plasmid vector (Addgene, Teddington, UK), which is a mammalian expression vector, to tag proteins with the 3×Flag epitope at the N-terminus, and pCMV-3Tag-2 (Addgene, Teddington, UK) to tag proteins with the 3×Myc epitope at the N-terminus. cDNA, which was reverse transcribed from RNA extracted from HCT116 cells using oligo-dT primers (Thermo Fisher Scientific, Waltham, MA, USA), was used as the template to amplify the coding sequences of target genes. The primer sets used are listed in Supplementary Table S1. The amplified target gene sequences were cloned into plasmid vectors using appropriate restriction enzymes (Enzynomics, Daejeon, Korea) and a DNA ligation kit (Takara Bio, Shiga, Japan) according to the manufacturers’ protocols. For site-directed/deletion mutagenesis, plasmids containing wild-type target genes were used as a template to amplify mutant target genes. The primers used for mutagenesis are listed in Supplementary Table S2. Wild-type and mutant ubiquitins cloned into the pRK5 vector (pRK5-HA-Ub WT (Addgene plasmid 17608), K0(17603), K6(22900), K11(22901), K27(22902), K29(22903), K33(17607), K48(17605), K63(17606)) were kindly provided by Drs. Ted Dawson and Sandra Weller.