Expression of wild type and mutant ELOVL4 in cell culture: subcellular localization and cell viability

Mol Vis. 2004 Mar 31:10:248-53.


Purpose: ELOVL4 is a member of the fatty acid elongase (ELO) family of genes. Mutations of this gene are responsible for autosomal dominant Stargardt-like macular degeneration. However, the specific role of ELOVL4 in photoreceptor cells and the mechanism by which mutations in ELOVL4 causes macular degeneration are not known. In this study we examined the subcellular localization of wild type (wt) and mutant (mt) ELOVL4 EGFP fusion protein and the potential functional consequence of mtELOVL4 expression on cell viability.

Methods: Wt and mt ELOVL4 were expressed as EGFP fusion proteins in NIH 3T3 and HEK293 cells. Subcellular localizations of the fusion proteins were determined with a series of organelle-specific markers for endoplasmic reticulum (pDsRed2-ER), mitochondria (pDsRed2-Mito), peroxisomes (pDsRed2-Peroxi), and Golgi (BODIPY TR). Transfected cells were viewed using confocal and episcopic-fluorescence microscopy. Western blot analysis was performed to assess protein expression using an anti-GFP antibody. TUNEL staining was used to quantify apoptotic cell death.

Results: In cell transfection studies, wtELOVL4/EGFP fusion protein localized preferentially to the endoplasmic reticulum (ER) and was not found to be discernibly present in mitochondria, peroxisomes, or Golgi. In contrast, the truncated mutant fusion protein (which has no ER retention signal) appeared to be mislocalized to other compartments within transfected cells. Transfected cells expressing mtELOVL4/EGFP fusion protein exhibited induction of apoptotic cell death.

Conclusions: Unlike wtELOVL4/EGFP fusion protein, the mtELOVL4/EGFP fusion protein did not localize to the ER but rather appeared to be sequestered elsewhere in an aggregated pattern in the cytoplasm. The apoptosis induced by the mutant ELOVL4 fusion protein may be the mechanism whereby photoreceptor cells degenerate in Stargardt-like macular degeneration. Our study has provided an important in vitro model system for further assessment of ELOVL4 biochemical functions.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Apoptosis*
  • Blotting, Western
  • Cell Survival
  • Cytoplasm / metabolism
  • Endoplasmic Reticulum / metabolism
  • Eye Proteins / genetics
  • Eye Proteins / metabolism*
  • Green Fluorescent Proteins
  • Humans
  • Immunoblotting
  • Kidney / cytology
  • Kidney / embryology
  • Kidney / metabolism
  • Luminescent Proteins / metabolism
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Mutation
  • NIH 3T3 Cells / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Subcellular Fractions
  • Transfection


  • ELOVL4 protein, human
  • Elovl4 protein, mouse
  • Eye Proteins
  • Luminescent Proteins
  • Membrane Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins

Associated data

  • OMIM/600110