Abstract
ELOVL4 (elongation of very long chain fatty acids 4) is a member of the ELO family of proteins involved in the biosynthesis of very long chain fatty acids. Protein truncation mutations in ELOVL4 have been identified in patients with autosomal dominant Stargardt-like macular degeneration. To determine whether a dominant negative mechanism is responsible for the autosomal dominant inheritance pattern of this disease, we studied the subcellular localization and interaction of wild type and mutant ELOVL4 in COS-7 and HEK 293T cultured cells by immunofluorescence and co-immunoprecipitation. Wild type ELOVL4 containing an endoplasmic reticulum retention sequence was localized to the endoplasmic reticulum as expected. In contrast, disease-associated C-terminal truncation ELOVL4 mutants accumulated as large inclusions exhibiting aggresome-like characteristics in a juxtanuclear position within COS-7 cells. When the wild type and mutant proteins were co-expressed incultured cells, wild type ELOVL4 co-purified with mutant ELOVL4 on an immunoaffinity column and co-localized with the mutant protein in aggresome-like inclusions adjacent to the nucleus. These results indicate that wild type and mutant ELOVL4 form a complex that exhibits an abnormal subcellular localization found for individually expressed mutant ELOVL4. From these studies, we conclude that disease-linked C-terminal truncation mutants of ELOVL4 exert a dominant negative effect on wild type ELOVL4, altering its subcellular localization. This dominant negative mechanism contributes to the autosomal dominant inheritance of Stargardt-like macular dystrophy.
Highlights
Common form of inherited early onset macular degeneration [1]
The gene responsible for two related forms of autosomal dominant macular degeneration known as Stargardt-like macular dystrophy (OMIM 600110) and autosomal dominant macular dystrophy (OMIM 600100) was recently shown to encode a membrane protein known as ELOVL4 for elongation of very long chain fatty acids 4 [9]
C-terminal truncation mutations in ELOVL4 have been identified in patients with an autosomal dominant form of macular degeneration known as Stargardt-like macular dystrophy; the mechanism underlying this retinal disease is presently unknown
Summary
Common form of inherited early onset macular degeneration [1]. This autosomal recessive disorder has been linked to mutations in ABCA4, an ATP-binding cassette transporter implicated in the removal of retinal derivatives from photoreceptor disc membranes [2,3,4]. Both antibodies labeled a 32-kDa protein in human retinal extracts and membranes from HEK 293T cells expressing the human ELOVL4 protein (Fig. 1A). To confirm that the 32-kDa protein is ELOVL4, membrane proteins from HEK 293T cells expressing the Myc-ELOVL4 protein were separated on SDS gels, and Western blots were labeled with the 9E10 antibody to detect the N-terminal Myc tag and the Elo 6F1 and Elo 8E4 antibodies (Fig. 1B).
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