Expression of CTNS alleles: subcellular localization and aminoglycoside correction in vitro.
Mutations in CTNS result in one of three forms of cystinosis: benign, intermediate, or nephropathic. Homozygosity for a nonsense mutation in CTNS (753G -->A), encoding a premature termination codon (PTC) at amino acid 138 (W138X), results in nephropathic cystinosis. Gentamicin is known to induce PTC readthrough and hence full-length protein production. We demonstrate that addition of gentamicin (300 microg/ml) to cystinotic fibroblasts leads to depletion of intracellular cystine in cell lines with a premature termination codon, but not in those with a large deletion or a deletion leading to a frameshift mutation. Plasmids were constructed with GFP as a C-terminal or N-terminal fusion to CTNS. The normal CTNS protein fused with either N- or C-terminal GFP colocalized with Lysotracker red, a fluorescent stain which selectively accumulates in lysosomes. PTC-GFP, a construct with GFP fused to the C-terminus of CTNS containing a PTC, allowed GFP to serve as a reporter of PTC readthrough. No significant fluorescence was observed in PTC-GFP-transfected cells in the absence of gentamicin but was seen and localized to lysosomes in its presence. A patient with a splice site mutation (IVS11 + 2T -->C) that eliminates the GYDQL lysosomal targeting sequence of cystinosin on one allele, and a PTC mutation (753G -->A) on the other, displays the intermediate phenotype. Transfection of the splice site mutant allele into CTNS null fibroblasts produced cystine depletion. Plasmids with GFP fused to the N-terminus of CTNS containing the splice site mutation (GFP-SS) were constructed. While the normal CTNS-GFP fusion protein was found to colocalize with Lysotracker red almost exclusively, the GFP-SS fusion product was found in the plasma membrane and cytoplasm, as well as lysosomes. A second lysosomal targeting motif in CTNS is present in this sequence, just proximal to the mutation, accounting for the partial lysosomal localization.
Helip-Wooley
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A
Hayward Human Genetics Center, Tulane University School of Medicine, 1430 Tulane Avenue SL/31, New Orleans, Louisiana 70112, USA. ahelip@tulane.edu
Park
M A
MA
Lemons
R M
RM
Thoene
J G
JG
D000483:Alleles; D026921:Amino Acid Transport Systems, Neutral; D000617:Aminoglycosides / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002478:Cells, Cultured; D018389:Codon, Nonsense / Q000235:genetics; D003554:Cystinosis / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005839:Gentamicins / Q000494:pharmacology; D006023:Glycoproteins; D006801:Humans; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics*; D026901:Membrane Transport Proteins; D017354:Point Mutation / Q000187:drug effects; D013347:Subcellular Fractions
D026921:Amino Acid Transport Systems, Neutral; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; C111445:CTNS protein, human; D018389:Codon, Nonsense; D005839:Gentamicins; D006023:Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins