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p53 and tumor necrosis factor alpha regulate the expression of a mitochondrial chloride channel protein
E Fernández-Salas1, M Sagar, C Cheng
1Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA. esterf@nih.gov
Abstract:
A novel chloride intracellular channel (CLIC) gene, clone mc3s5/mtCLIC, has been identified from differential display analysis of differentiating mouse keratinocytes from p53+/+ and p53-/- mice. The 4.2-kilobase pair cDNA contains an open reading frame of 762 base pairs encoding a 253-amino acid protein with two putative transmembrane domains. mc3s5/mtCLIC protein shares extensive homology with a family of intracellular organelle chloride channels but is the first shown to be differentially regulated. mc3s5/mtCLIC mRNA is expressed to the greatest extent in vivo in heart, lung, liver, kidney, and skin, with reduced levels in some organs from p53-/- mice. mc3s5/mtCLIC mRNA and protein are higher in p53+/+ compared with p53-/- basal keratinocytes in culture, and both increase in differentiating keratinocytes independent of genotype. Overexpression of p53 in keratinocytes induces mc3s5/mtCLIC mRNA and protein. Exogenous human recombinant tumor necrosis factor alpha also up-regulates mc3s5/mtCLIC mRNA and protein in keratinocytes. Subcellular fractionation of keratinocytes indicates that both the green fluorescent protein-mc3s5 fusion protein and the endogenous mc3s5/mtCLIC are localized to the cytoplasm and mitochondria. Similarly, mc3s5/mtCLIC was localized to mitochondria and cytoplasmic fractions of rat liver homogenates. Furthermore, mc3s5/mtCLIC colocalized with cytochrome oxidase in keratinocyte mitochondria by immunofluorescence and was also detected in the cytoplasmic compartment. Sucrose gradient-purified mitochondria from rat liver confirmed this mitochondrial localization. This represents the first report of localization of a CLIC type chloride channel in mitochondria and the first indication that expression of an organellular chloride channel can be regulated by p53 and tumor necrosis factor alpha.
Insights
Researchers discovered a new chloride intracellular channel (CLIC) gene, mc3s5/mtCLIC, in mouse cells. This gene
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Chloride intracellular channels (CLICs) are a family of organelle ion channels.
- The regulation and precise localization of CLIC family members are not fully understood.
- The role of the p53 tumor suppressor in regulating cellular processes is well-established.
Purpose of the Study:
- To identify and characterize a novel CLIC gene in mouse keratinocytes.
- To investigate the differential regulation of this new CLIC gene.
- To determine the subcellular localization of the novel CLIC protein.
Main Methods:
- Differential display analysis of mouse keratinocytes from p53+/+ and p53-/- mice.
- cDNA cloning and sequencing to identify the gene and protein structure.
- Quantitative analysis of mRNA and protein expression levels.
- Subcellular fractionation and immunofluorescence microscopy for protein localization.
Main Results:
- A novel CLIC gene, mc3s5/mtCLIC, was identified and characterized.
- mc3s5/mtCLIC expression is differentially regulated by p53 and tumor necrosis factor alpha.
- The mc3s5/mtCLIC protein is localized to the cytoplasm and mitochondria.
- This is the first CLIC channel reported to be localized in mitochondria.
Conclusions:
- mc3s5/mtCLIC represents a novel, differentially regulated CLIC channel.
- Its mitochondrial localization suggests a role in mitochondrial function.
- p53 and TNF-alpha are regulators of this organellar chloride channel's expression.