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Unexpected intracellular localization of the AMD-associated cystatin C variant.
- Source :
-
Traffic (Copenhagen, Denmark) [Traffic] 2004 Nov; Vol. 5 (11), pp. 884-95. - Publication Year :
- 2004
-
Abstract
- Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.
- Subjects :
- Aging
Alanine chemistry
Blotting, Western
Cell Nucleus metabolism
Cells, Cultured
Cystatins genetics
Cystatins metabolism
Cytoplasm metabolism
DNA Primers chemistry
Electrophoresis, Polyacrylamide Gel
Golgi Apparatus metabolism
Green Fluorescent Proteins metabolism
HeLa Cells
Humans
Microscopy, Confocal
Microscopy, Fluorescence
Mitochondria metabolism
Mitochondria pathology
Mutagenesis, Site-Directed
Mutation
Pigment Epithelium of Eye cytology
Plant Proteins
Plasmids metabolism
Protein Structure, Tertiary
Threonine chemistry
Time Factors
Transfection
Trypsin pharmacology
Cystatins biosynthesis
Macular Degeneration genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1398-9219
- Volume :
- 5
- Issue :
- 11
- Database :
- MEDLINE
- Journal :
- Traffic (Copenhagen, Denmark)
- Publication Type :
- Academic Journal
- Accession number :
- 15479453
- Full Text :
- https://doi.org/10.1111/j.1600-0854.2004.00230.x