A Novel Golgi Localization Signal RPWS For Tumor Suppressor UBIAD1

UBIAD1is involved in dyslipimedia-induced SCD (Schnyder crystalline cornealdystrophy), Parkinson’s disease, multiple human cancers and vitamin K biosynthesis.Currently the subcellular localization of UBIAD1is not clear. Since UBIAD1is a tumorsuppressor for bladder carcinoma, we studied the subcellular localization of UBIAD1inHEK293cell line and bladder carcinoma cells J82and T24cell lines.In order to study the UBIAD1subcellular localization, we used a combination ofbiochemical and cell biology approaches. First we constructed the expression vectorpEGFP-N1containing full length UBIAD1gene, truncated of UBIAD1gene, mutantedUBIAD1gene. Then we transfected the cell line HEK293with those vectors. Observingthe green fluorescent protein by confocal microscope, we determined the subcellularlocalization and traffings of UBIAD1. Over-expression results are consistent withimmunohistochemical results. Live cell fluorescence imaging results indicate thatUBIAD1is co-localized with Golgi apparatus, which is the first report for Golgilocalization of UBIAD1. The N-terminus of UBIAD1is vital for its subcellularlocalization, since the N-terminus of UBIAD1can targeted a plasma membrane proteinorail1to Golgi.Bioinformatic analysis suggests that the Golgi retention signal for UBIAD1is theresidues on the54-57amino acides of UBIAD1N-terminus RPWS motif. Through aseries of point mutation analysis，we tested and verified the hypothesis. The RPWS motifis the golgi localization signal affecting UBIAD1protein retention on the Golgi， whichforms an Arginine finger on the N terminus of UBIAD1.In order to study the intracellular trafficking of UBIAD1，the HKE293cell line weretreated with BFA which affects the UBIAD1retention on the Golgi, indicating theUBIAD1transport from the endoplasmic reticulum (ER) to the Golgi via aCOPII-mediated mechanism.Aim to check whether the RPWS motif affects UBIAD1tumor suppressing activity,flow cytometry was performed to analyze the cell apoptosis of bladder carcinoma T24 cells by UBIAD1and UBIAD1mutant RPWSâ†'AAAA protein.Compared with wild typeUBIAD1, the apoptosis affect of mutanted UBIAD1protein decreases，indicating thatproper Golgi localization of UBIAD1is vital for its tumor suppressing activity.