title: Quaternary structure assessment of ICln by fluorescence resonance energy transfer (FRET) in vivo.
abstract: ICln is a ubiquitously expressed multifunctional protein that plays a critical role in regulatory volume decrease after cell swelling. The majority of ICln is localized in the cytosol and a small fraction of ICln associates with the plasma membrane. In artificial lipid bilayers ICln forms ion channels, and a putative channel model predicts the association of at least two ICln molecules to form a functional ion-conducting pore. Oligomers of ICln have been demonstrated in cytosolic fractions of different cells by native PAGE and gel filtration analysis, but these data have not yet been verified in vivo, and the basis of ICln homooligomerization is unknown. In silico prediction of the quaternary structure of ICln from its primary structure predicts that ICln forms a dimer, and that the C-terminus of ICln may be essential for the intermolecular interaction. To explore the quaternary structure of ICln in living NIH3T3 fibroblasts, we performed fluorescence resonance energy transfer (FRET) experiments using eCFP (donor) and eYFP (acceptor) fused to the C- and/or N-termini of both full length wild type ICln and of C-terminal truncation mutants thereof (ICln(159) and ICln(134)). FRET was assessed by the acceptor photobleaching technique. Here we show that ICln forms oligomers in vivo, and demonstrate intermolecular FRET between the C-, but not the N-termini of full length ICln. In the truncation mutant ICln(159) oligomerization occurs and intermolecular FRET between N-termini can be detected, which indicates that the C-terminus of ICln sterically interferes with interactions between N-termini in full length ICln oligomers. In cells expressing the truncation mutant ICln(134) no FRET between C- and/or N-termini could be measured, suggesting the absence of interaction and a role of amino acids P135-Q159 in the oligomerization of ICln.
authors.affiliation: Institute of Physiology and Pathophysiology, Paracelsus Medical University, Salzburg, Austria.
authors.forename: Sabine
authors.initials: S
authors.lastname: Schmidt
authors.forename: Martin
authors.initials: M
authors.lastname: Jakab
authors.forename: Ivano
authors.initials: I
authors.lastname: Costa
authors.forename: Johannes
authors.initials: J
authors.lastname: Fürst
authors.forename: Andrea
authors.initials: A
authors.lastname: Ravasio
authors.forename: Markus
authors.initials: M
authors.lastname: Paulmichl
authors.forename: Guido
authors.initials: G
authors.lastname: Botta
authors.forename: Markus
authors.initials: M
authors.lastname: Ritter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D041681:NIH 3T3 Cells; D011485:Protein Binding; D020836:Protein Structure, Quaternary
chemical_list: D007473:Ion Channels; D008164:Luminescent Proteins; D050505:Mutant Proteins; C527701:enhanced cyan fluorescent protein; D049452:Green Fluorescent Proteins