Stargardt-like macular dystrophy protein ELOVL4 exerts a dominant negative effect by recruiting wild-type protein into aggresomes.
OBJECTIVE
Mutations in the gene Elongation of very long-chain fatty acids-4 (ELOVL4) have been shown to be associated with autosomal dominant Stargardt-like macular dystrophy (STGD3). ELOVL4 is expressed in photoreceptors and encodes a putative transmembrane protein of 314 amino acids with an endoplasmic reticulum (ER) retention signal. A 5 bp deletion in exon 6 of ELOVL4 observed in some STGD3 patients results in the truncation of the protein and loss of the ER retention signal. To understand the disease mechanism underlying STGD3 we studied the intracellular trafficking of the wild-type and a 5 bp deletion mutant of ELOVL4.


METHODS
Wild-type and mutant ELOVL4 proteins with the N-terminal GFP/V5 tags were expressed in COS-7 cells. Expression and the intracellular localization of the wild-type and mutant proteins were characterized by immunocytochemistry and western blot analysis using tag- and organelle-specific antibodies. Interaction between the wild-type and mutant proteins was studied by two-dimensional gel electrophoresis and fluorescence resonance energy transfer (FRET) analysis.


RESULTS
The mutant ELOVL4 protein exerted a dominant negative effect when the wild-type and 5 bp deletion mutant ELOVL4 proteins were co-expressed in COS-7 cells. Immunocytochemical analysis, two-dimensional gel electrophoresis and FRET revealed that the mutant ELOVL4 interacts with the wild-type protein, forming higher molecular mass complexes that accumulate in aggresomes.


CONCLUSIONS
In the presence of mutant ELOVL4 protein, the wild-type protein was recruited into perinuclear cytoplasmic inclusions that resemble aggresomes. The interaction between the wild-type and mutant forms of ELOVL4 and the resultant alteration in the trafficking of the wild-type ELOVL4 protein suggest a mechanism for the pathogenicity observed in patients with autosomal dominant STGD3.
Vasireddy
Vidyullatha
V
Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI, USA.
Vijayasarathy
Camasamudram
C
Huang
Jibiao
J
Wang
Xiaofei F
XF
Jablonski
Monica M
MM
Petty
Howard R
HR
Sieving
Paul A
PA
Ayyagari
Radha
R
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells / Q000378:metabolism; D018929:Cell Culture Techniques; D002522:Chlorocebus aethiops; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D017353:Gene Deletion; D015870:Gene Expression; D005822:Genetic Vectors; D006056:Golgi Apparatus / Q000378:metabolism; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
C418975:ELOVL4 protein, human; D005136:Eye Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014746:Vimentin