Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Dec 15;360(Pt 3):645-9.
doi: 10.1042/0264-6021:3600645.

Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells

R K Jain  1 , P B Joyce, M Molinete, P A Halban, S U Gorr
Affiliations

Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells

R K Jain et al. Biochem J. .

Abstract

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 27 kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys(49) and Cys(71)), which could play a role in this oligomerization. Site-directed mutagenesis of Cys(49) and Cys(71) 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.

PubMed Disclaimer

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Nat Biotechnol. 1996 Oct;14(10):1246-51 - PubMed
    1. Nature. 1970 Aug 15;227(5259):680-5 - PubMed
    1. Nucleic Acids Res. 2000 Jan 1;28(1):235-42 - PubMed
    1. Endocrinology. 2001 Feb;142(2):864-73 - PubMed
    1. Biochim Biophys Acta. 1999 Mar 9;1410(3):287-98 - PubMed

Publication types

MeSH terms