Abstract
Evidence was recently reported that the cysteine proteinase inhibitor, cystatin C, is highly expressed by cultured human retinal pigment epithelial (RPE) cells. As a step towards understanding possible functions of this protein associated with the RPE, the localization, targetting and trafficking of cystatin C were investigated. Constructs encoding an enhanced variant of green fluorescent protein (EGFP) fused to precursor cystatin C and to mature cystatin C were made and transfected into cultured human RPE cells. Expression of fusion proteins was monitored in vivo by fluorescence confocal microscopy. In cells transfected with precursor cystatin C-EGFP, fluorescence was initially targetted to the perinuclear zone, co-localizing with the Golgi apparatus. Transfected cells were observed at intervals over a period of up to 3 weeks, during which time fluorescent vesicles developed peripherally and basally while fluorescence continued to be detected in the Golgi region. Immunochemical analysis of cell lysates confirmed the expression of a fusion protein recognized by antibodies to both cystatin C and EGFP. Cells transfected with the construct lacking the leader peptide of precursor cystatin C presented a diffuse and weak fluorescence. Together, these results imply a leader sequence-dependent processing of cystatin C through the secretory pathway of RPE cells. This was confirmed by the detection, by Western blotting, of the chimaeric protein alongside endogenous cystatin C in the medium of transfected RPE cells.
Original language | English |
---|---|
Pages (from-to) | 229-36 |
Number of pages | 8 |
Journal | Molecular Membrane Biology |
Volume | 18 |
Issue number | 3 |
DOIs | |
Publication status | Published - 30 Oct 2001 |
Keywords
- Amino Acid Sequence
- Blotting, Western
- Cells, Cultured
- Cystatin C
- Cystatins/chemistry
- Golgi Apparatus/metabolism
- Green Fluorescent Proteins
- HeLa Cells
- Humans
- Luminescent Proteins/metabolism
- Microscopy, Fluorescence
- Molecular Weight
- Pigment Epithelium of Eye/cytology
- Protein Precursors/chemistry
- Protein Processing, Post-Translational
- Protein Transport
- Time Factors
- Transfection