Unexpected intracellular localization of the AMD-associated cystatin C variant.
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.
Unit of Ophthalmology, Department of Medicine, Centre for Cell Imaging, School of Biological Sciences, University of Liverpool, UK. lparaoan@liv.ac.uk
Luminita
L
Paraoan
Arjuna
A
Ratnayaka
Dave G
DG
Spiller
Paul
P
Hiscott
Michael R H
MR
White
Ian
I
Grierson
D000375:Aging; D000409:Alanine / Q000737:chemistry; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D015891:Cystatins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008268:Macular Degeneration / Q000235:genetics*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010857:Pigment Epithelium of Eye / Q000166:cytology; D010940:Plant Proteins; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
D015891:Cystatins; D017931:DNA Primers; D010940:Plant Proteins; C116405:cystatin protein, Castanea sativa; D049452:Green Fluorescent Proteins; D013912:Threonine; D014357:Trypsin; D000409:Alanine