Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.653215
Title: Rat brain chloride intracellular channel protein CLIC4 : subcellular localisation and protein interaction studies
Author: Karoulias, Nikolaos
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2002
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
Abstract:
Rat brain CLIC4 (p64H1) is a member of a family of recently identified chloride intracellular channel (CLIC) proteins that share homology with the putative bovine anion channel protein p64. CLIC comprises a 28 kDa protein, with a large cytoplasmic domain, a single transmembrane domain and a small intraluminal domain. Similar to other CLIC proteins, CLIC4 shows a wide tissue distribution with an unusual dual localisation in both membrane and cytoplasmic fractions. However, the molecular bases of its cellular localisation, and anion channel activity are unclear. To help address these problems, this study set out to identify CLIC4-associated proteins. Recombinant full-length and truncated CLIC4 were expressed in bacteria as glutathione-S-transferase (GST) fusion proteins, and purified by glutathione affinity chromatography. In “pull-down” assays, several rat brain cytosol proteins were found to bind directly or indirectly to the cytoplasmic domain of CLIC4, including dynamin I, β-actin, tubulin and 14-3-3 isoforms. These interactions were confirmed in vivo by immunoprecipitation. CLIC4 was over-expressed in cultured mammalian cells as full-length and Flag-tagged fusion proteins. The subcellular localisation of native and heterologously expressed CLIC4 was investigated by indirect immunofluorescence. Biochemical and immunofluorescence analyses of CLIC4-transfected cells demonstrated partial co-localisation of CLIC4 with caveolin, and with functional caveolae. Several consensus phosphorylation sites have previously been identified in the CLIC4 protein sequence. CLIC4 interactions with protein kinases were confirmed by demonstrating phosphorylation by protein kinase C, protein kinase A and tyrosine kinase in vitro. The incorporation of purified recombinant CLIC4 into planar lipid bilayers failed to demonstrate specific anion channel activity, suggesting that interactions with some of the identified cellular proteins might underlie the anion channel activity of CLIC4.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.653215  DOI: Not available
Share: