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

[摘要] Purpose: ELOVL4 is a member of the fatty acid elongase (ELO)family of genes. Mutations of this gene are responsible for autosomaldominant Stargardt-like macular degeneration. However, the specific roleof ELOVL4 in photoreceptor cells and the mechanism by which mutations inELOVL4 causes macular degeneration are not known. In this study weexamined the subcellular localization of wild type (wt) and mutant (mt)ELOVL4 EGFP fusion protein and the potential functional consequence ofmtELOVL4 expression on cell viability.Methods: Wt and mt ELOVL4 were expressed as EGFP fusion proteins inNIH 3T3 and HEK293 cells. Subcellular localizations of the fusionproteins were determined with a series of organelle-specific markers forendoplasmic reticulum (pDsRed2-ER), mitochondria (pDsRed2-Mito),peroxisomes (pDsRed2-Peroxi), and Golgi (BODIPY TR). Transfected cellswere viewed using confocal and episcopic-fluorescence microscopy.Western blot analysis was performed to assess protein expression usingan anti-GFP antibody. TUNEL staining was used to quantify apoptotic celldeath.Results: In cell transfection studies, wtELOVL4/EGFP fusion proteinlocalized preferentially to the endoplasmic reticulum (ER) and was notfound to be discernibly present in mitochondria, peroxisomes, or Golgi.In contrast, the truncated mutant fusion protein (which has no ERretention signal) appeared to be mislocalized to other compartmentswithin transfected cells. Transfected cells expressing mtELOVL4/EGFPfusion protein exhibited induction of apoptotic cell death.Conclusions: Unlike wtELOVL4/EGFP fusion protein, the mtELOVL4/EGFPfusion protein did not localize to the ER but rather appeared to besequestered elsewhere in an aggregated pattern in the cytoplasm. Theapoptosis induced by the mutant ELOVL4 fusion protein may be themechanism whereby photoreceptor cells degenerate in Stargardt-likemacular degeneration. Our study has provided an important in vitro modelsystem for further assessment of ELOVL4 biochemical functions.

[学科分类] 生物化学/生物物理