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Unexpected intracellular localization of the AMD-associated cystatin C variant

Unexpected intracellular localization of the AMD-associated cystatin C variant
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.

Aging, Alanine, Blotting, Western, Cell Nucleus, Cells, Cultured, Cystatins, Cytoplasm, DNA Primers, Electrophoresis, Polyacrylamide Gel, Golgi Apparatus, Green Fluorescent Proteins, HeLa Cells, Humans, Macular Degeneration, Microscopy, Confocal, Microscopy, Fluorescence, Mitochondria, Mutagenesis, Site-Directed, Mutation, Pigment Epithelium of Eye, Plant Proteins, Plasmids, Protein Structure, Tertiary, Threonine, Time Factors, Transfection, Trypsin, Journal Article, Research Support, Non-U.S. Gov't
1398-9219
884-95
Paraoan, Luminita
252bc6c9-e9b1-4bbb-a2d3-d7fb91826b0e
Ratnayaka, Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd
Spiller, Dave G
e2d4ae55-26f1-435f-88b7-cded8d3ebc56
Hiscott, Paul
c5f18179-36ab-4466-98a3-906e6638e10a
White, Michael R H
d0d97c08-20c6-4452-a7f4-db92ae05109c
Grierson, Ian
b7edfcbe-f96e-4fe9-a5f1-b235300d011a
Paraoan, Luminita
252bc6c9-e9b1-4bbb-a2d3-d7fb91826b0e
Ratnayaka, Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd
Spiller, Dave G
e2d4ae55-26f1-435f-88b7-cded8d3ebc56
Hiscott, Paul
c5f18179-36ab-4466-98a3-906e6638e10a
White, Michael R H
d0d97c08-20c6-4452-a7f4-db92ae05109c
Grierson, Ian
b7edfcbe-f96e-4fe9-a5f1-b235300d011a

Paraoan, Luminita, Ratnayaka, Arjuna, Spiller, Dave G, Hiscott, Paul, White, Michael R H and Grierson, Ian (2004) Unexpected intracellular localization of the AMD-associated cystatin C variant. Traffic, 5 (11), 884-95. (doi:10.1111/j.1600-0854.2004.00230.x).

Record type: Article

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.

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More information

e-pub ahead of print date: 14 September 2004
Published date: November 2004
Keywords: Aging, Alanine, Blotting, Western, Cell Nucleus, Cells, Cultured, Cystatins, Cytoplasm, DNA Primers, Electrophoresis, Polyacrylamide Gel, Golgi Apparatus, Green Fluorescent Proteins, HeLa Cells, Humans, Macular Degeneration, Microscopy, Confocal, Microscopy, Fluorescence, Mitochondria, Mutagenesis, Site-Directed, Mutation, Pigment Epithelium of Eye, Plant Proteins, Plasmids, Protein Structure, Tertiary, Threonine, Time Factors, Transfection, Trypsin, Journal Article, Research Support, Non-U.S. Gov't

Identifiers

Local EPrints ID: 422428
URI: http://eprints.soton.ac.uk/id/eprint/422428
ISSN: 1398-9219
PURE UUID: 5decfc66-12b8-4130-8a65-9df46b38ca6c
ORCID for Arjuna Ratnayaka: ORCID iD orcid.org/0000-0002-1027-6938

Catalogue record

Date deposited: 24 Jul 2018 16:30
Last modified: 10 Nov 2021 03:34

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Contributors

Author: Luminita Paraoan
Author: Dave G Spiller
Author: Paul Hiscott
Author: Michael R H White
Author: Ian Grierson

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