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.
Highlights
From the Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI and the ¶Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892
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
Sucrose gradient-purified mitochondria from rat liver confirmed this mitochondrial localization. This represents the first report of localization of a chloride intracellular channel (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 ␣
Summary
Vol 274, No 51, Issue of December 17, pp. 36488 –36497, 1999 Printed in U.S.A. p53 and Tumor Necrosis Factor ␣ Regulate the Expression of a Mitochondrial Chloride Channel Protein*. 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 ␣. Deletion of one or both p53 alleles enhances the establishment of immortal keratinocyte cell lines in vitro [10], suggesting that p53 could participate in keratinocyte mortality in a more subtle way, not involving expression of the major differentiation-related markers To detect such a contribution of p53 to keratinocyte differentiation or mortality, differential expression of mRNA sequences was examined in keratinocytes isolated from p53 wild-type and null mice and induced to differentiate by Ca2ϩ in vitro, a condition known to increase p53-dependent transcriptional activity. By sequence homology and subcellular localization studies, we found that mc3s5/mtCLIC is a chloride channel protein found in the cytoplasm and mitochondria
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
