p53 and tumor necrosis factor alpha regulate the expression of a mitochondrial chloride channel protein.
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.
Fernández-Salas
E
E
Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA. esterf@nih.gov
Sagar
M
M
Cheng
C
C
Yuspa
S H
SH
Weinberg
W C
WC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018118:Chloride Channels / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D016158:Genes, p53; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008928:Mitochondria / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
D018118:Chloride Channels; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53