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The Translocation Of CLIC5A To Membranes As A Peripheral Protein

  • Author / Creator
    Kim, Jong Suck
  • Chloride intracellular channel (CLIC) proteins comprise six members CLIC1–CLIC6 in mammals and mediate functions not fully known. Their founding member was isolated from bovine kidney using the chloride channel inhibitor indanyloxyacetic acid-94, so CLICs were assumed to be typical anion channels. However, CLIC proteins possess several properties discordant with classical channels, and there are doubts regarding their capacity to form the integral transmembrane (TM) architecture of α- and β-type ion channels. Chief among such properties are their structural dimorphism. CLICs assume multiple conformational folds, and the transitions between them accompany their translocation to their diverse subcellular locales. Though several X-ray structures of their soluble form have been solved, the signaling pathways which mediate their transition in cells have not yet been identified, nor has any membrane-associated structure been solved.

    In comparison to the diverse expression of most CLICs, CLIC5A is highly enriched in glomerular tissue within podocytes and endothelial cells and is frequently found in actin-rich projections of the plasma membrane (PM) in a highly polarized distribution. At the apical PM, CLIC5A is localized in clusters containing phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2), where it promotes PM–actin cytoskeleton linkages by stimulating the activation of the cross-linking ezrin-radixin-moesin (ERM) proteins, in part by Rac1-induced activation of phosphoinositide kinases. Given that these signaling events are centralized at and near the PM, it is important to discern the cellular processes translocating CLIC5A from the cytosol to this site.

    The primary focus of this thesis is to investigate the translocation process of CLIC5A using structural and cellular approaches, and to challenge widely-held notions that CLICs form TM channels. NMR spectra of truncated N-terminal CLIC5A mutants containing the putative transmembrane domain were obtained to reveal that CLIC5A adopts multiple conformations in solution. Next, CLIC5A was assayed in transfected COS-7 cells under membrane permeabilizing and intact conditions, which revealed that this protein was detected only in the former condition, strongly suggesting CLIC5A is confined in the intracellular space with no appreciable extracellular domains.

    Furthermore, subcellular fractions extracted by differential detergent fractionation revealed that overexpressed CLIC1, CLIC4, and CLIC5A were predominantly cytosolic and not detectable in membrane fractions. However, Calyculin A-induced phosphatase inhibition significantly increased the retrieval of CLIC4, CLIC5A, and to a much lesser extent CLIC1, in membrane fractions. For CLIC5A, the phosphorylation-driven translocation to membranes was sensitive to Staurosporine, suggesting a protein kinase C (PKC) as a likely candidate mediating this process. Multifactorial prediction analyses reveal that CLIC5A is likely to be phosphorylated at two C-terminal Thr residues during this process.

    Overall, this study demonstrates for the first time that CLIC4 and CLIC5A are translocated to membranes in a phosphorylation-driven process which for CLIC5A is mediated by a PKC. Further, our results corroborate earlier claims that CLIC5A does not form an integral TM protein and thus unlikely to function as a classical ion channel.

  • Subjects / Keywords
  • Graduation date
    Fall 2018
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/R33T9DP2C
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.