Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys49 and Cys71), which could play a role in this oligomerization. Site-directed mutagenesis of Cys49 and Cys71 showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.
Skip Nav Destination
Article navigation
December 2001
- PDF Icon PDF LinkFront Matter
Research Article|
December 10 2001
Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells
Renu K. JAIN;
Renu K. JAIN
∗Department of Molecular, Cellular and Craniofacial Biology, University of Louisville, 501 South Preston Street, Louisville, KY 40292 U.S.A.
Search for other works by this author on:
Paul B. M. JOYCE;
Paul B. M. JOYCE
†Department of Chemistry and Biochemistry and Centre for Structural and Functional Genomics, Concordia University, 1455 de Maisonneuve Boulevard, Montreal, QC, H3G 1M8, Canada
Search for other works by this author on:
Miguel MOLINETE;
Miguel MOLINETE
‡Louis-Jeantet Research Laboratories, University Medical Center, 1211 Geneva 4, Switzerland
Search for other works by this author on:
Philippe A. HALBAN;
Philippe A. HALBAN
‡Louis-Jeantet Research Laboratories, University Medical Center, 1211 Geneva 4, Switzerland
Search for other works by this author on:
Sven-Ulrik GORR
Sven-Ulrik GORR
1
∗Department of Molecular, Cellular and Craniofacial Biology, University of Louisville, 501 South Preston Street, Louisville, KY 40292 U.S.A.
§Department of Biochemistry and Molecular Biology, University of Louisville, 501 South Preston Street, Louisville, KY 40292 U.S.A.
1To whom correspondence should be addressed (e-mail sven.gorr@louisville.edu).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
July 06 2001
Revision Received:
August 21 2001
Accepted:
October 10 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2001
2001
Biochem J (2001) 360 (3): 645–649.
Article history
Received:
July 06 2001
Revision Received:
August 21 2001
Accepted:
October 10 2001
Citation
Renu K. JAIN, Paul B. M. JOYCE, Miguel MOLINETE, Philippe A. HALBAN, Sven-Ulrik GORR; Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells. Biochem J 15 December 2001; 360 (3): 645–649. doi: https://doi.org/10.1042/bj3600645
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.