A novel Golgi retention signal RPWS for tumor suppressor UBIAD1

PLoS One. 2013 Aug 19;8(8):e72015. doi: 10.1371/journal.pone.0072015. eCollection 2013.

Abstract

UBIAD1 plays critical roles in physiology including vitamin K and CoQ10 biosynthesis as well as pathophysiology including dyslipimedia-induced SCD (Schnyder's corneal dystrophy), Parkinson's disease, cardiovascular disease and bladder carcinoma. Since the subcellular localization of UBIAD1 varies in different cell types, characterization of the exact subcellular localization of UBIAD1 in specific human disease is vital for understanding its molecular mechanism. As UBIAD1 suppresses bladder carcinoma, we studied its subcellular localization in human bladder carcinoma cell line T24. Since fluorescent images of UBIAD1-EGFP in T24, human prostate cancer cell line PC-3, human embryonic kidney cell line HEK293 and human hepatocyte cell line L02 are similar, these four cell lines were used for present study. Using a combination of fluorescent microscopy and immunohistochemistry, it was found that UBIAD1 localized on the Golgi and endoplasmic reticulum (ER), but not on the plasma membrane, of T24 and HEK293 cells. Using scanning electron microscopy and western blot analysis, we found that UBIAD1 is enriched in the Golgi fraction extracted from the L02 cells, verifying the Golgi localization of UBAID1. Site-directed mutagenesis showed that the RPWS motif, which forms an Arginine finger on the UBIAD1 N terminus, serves as the Golgi retention signal. With both cycloheximide and brefeldin A inhibition assays, it was shown that UBIAD1 may be transported from the endoplasmic reticulum (ER) to the Golgi by a COPII-mediated mechanism. Based upon flow cytometry analysis, it is shown that mutation of the RPWS motif reduced the UBIAD1-induced apoptosis of T24 cells, indicating that the proper Golgi localization of UBIAD1 influences its tumor suppressant activity. This study paves the way for further understanding the molecular mechanism of UBIAD1 in human diseases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Cell Line, Tumor
  • Dimethylallyltranstransferase / chemistry
  • Dimethylallyltranstransferase / genetics
  • Dimethylallyltranstransferase / metabolism*
  • Endoplasmic Reticulum / enzymology
  • Genes, Tumor Suppressor
  • Golgi Apparatus / enzymology*
  • HEK293 Cells
  • Humans
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Protein Sorting Signals
  • Protein Transport

Substances

  • Protein Sorting Signals
  • Dimethylallyltranstransferase
  • UBIAD1 protein, human

Grant support

This project was supported in part by the Grants from NSFC (P.R. China, No. 30971608, No. 81272210), 863 Grant of Ministry of Science and Technology (P.R. China, No. 2007AA09Z449), NSF of the Hubei Province (P.R. China, 2009CDB074), The Specific Key Project of Novel Medicine Discovery (P.R. China, 2009ZX09301-014) and International Collaboration Programs of Wuhan Science and Technology Bureau (P.R. China, No. 201070934334). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.