title: Loss of ER retention and sequestration of the wild-type ELOVL4 by Stargardt disease dominant negative mutants.
abstract: OBJECTIVE
  Mutations in ELOVL4, a member of the fatty acid elongase (ELO) family, are responsible for autosomal dominant Stargardt-like macular degeneration. The specific role of ELOVL4 in photoreceptors and the degenerative events induced by dominant ELOVL4 mutations are not well understood. As a first step to identifying possible mechanisms contributing to cellular dysfunction, we transfected HEK293 and COS cells with fluorescent-labeled wild-type and mutant ELOVL4 constructs. Effects of mutant ELOVL4 on interaction with wild-type protein were examined in this in vitro model.
  
  
  METHODS
  Wild-type and mutant ELOVL4 proteins including ELOVL4 truncation (270X, a truncated ELOVL4 protein at amino acid position 270) and ELOVL4 5 bp deletion (5bp-del) and ELOVL4 (5A, substituting the ER retention signal, KAKGD, with a five alanine amino acid tract) were expressed as EGFP or DsRed fusion proteins. Cellular localization of these proteins was examined by fluorescence microscopy. ELOVL4 protein aggregates were measured by co-immunoprecipitation and by sucrose gradient centrifugation followed by immunodetection with western blots. To study cellular status of cells expressing mutant ELOVL4 proteins, transfected cells were examined for upregulation of Bip and CHOP, markers for the unfolded protein response (UPR) by western blotting.
  
  
  RESULTS
  ELOVL4 mutants were not retained within the ER but were rather mislocalized and formed aggregates. Importantly, when cotransfected with wild-type ELOVL4, the mutants bound to and sequestered the wild-type protein into the aggregates. Expression of ELOVL4 mutants also induced UPR as evidenced by Bip and CHOP expression.
  
  
  CONCLUSIONS
  Using this in vitro cell system, we have identified alterations in wild-type ELOVL4 protein localization, aggregate formation, and the induction of cellular stress by the ELOVL4 mutants. We propose that "inactivation" of the wild-type ELOVL4 protein through sequestration to a non-ER compartment by ELOVL4 mutants may play a role in cellular dysfunction.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Eccles Institute of Human Genetics, University of Utah Health Sciences Center, Salt Lake City, UT, USA. gkaran@hmbg.utah.edu
authors.forename: Goutam
authors.initials: G
authors.lastname: Karan
authors.forename: Zhenglin
authors.initials: Z
authors.lastname: Yang
authors.forename: Kimberly
authors.initials: K
authors.lastname: Howes
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhao
authors.forename: Yali
authors.initials: Y
authors.lastname: Chen
authors.forename: D Josh
authors.initials: DJ
authors.lastname: Cameron
authors.forename: Yin
authors.initials: Y
authors.lastname: Lin
authors.forename: Erik
authors.initials: E
authors.lastname: Pearson
authors.forename: Kang
authors.initials: K
authors.lastname: Zhang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000378:metabolism; D008268:Macular Degeneration / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D018832:Molecular Chaperones / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051742:Transcription Factor CHOP / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation
chemical_list: C497249:DDIT3 protein, human; C418975:ELOVL4 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D005136:Eye Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D051742:Transcription Factor CHOP