Jain R K, Joyce P B, Molinete M, Halban P A, Gorr S U
Department of Molecular, Cellular and Craniofacial Biology, University of Louisville, 501 South Preston Street, Louisville, KY 40292, U.S.A.
Biochem J. 2001 Dec 15;360(Pt 3):645-9. doi: 10.1042/0264-6021:3600645.
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.
绿色荧光蛋白（GFP）被广泛用作报告蛋白，以监测细胞过程，包括细胞内蛋白质运输和分泌。一般来说，这种方法依赖于GFP作为一种被动报告蛋白。然而，最近有人指出，GFP在内分泌细胞的分泌途径中会发生寡聚化。为了表征这种寡聚化及其在GFP运输中的潜在作用，在GH4C1和AtT-20内分泌细胞中表达了增强型GFP（EGFP）的胞质形式和分泌形式。生化分析表明，胞质EGFP以27 kDa的单体形式存在，而EGFP的分泌形式形成了二硫键连接的寡聚体。EGFP含有两个半胱氨酸残基（Cys(49)和Cys(71)），它们可能在这种寡聚化中发挥作用。对Cys(49)和Cys(71)进行定点诱变表明，两个半胱氨酸残基都参与了二硫键相互作用。替换任何一个半胱氨酸残基都会导致寡聚体减少或消失，尽管EGFP分泌形式的二聚体仍然存在。这些残基的突变不会对EGFP的荧光产生不利影响。EGFP寡聚体储存在分泌颗粒中，并通过内分泌AtT-20细胞中的调节性分泌途径分泌。同样，EGFP的二聚体突变形式仍然通过调节性分泌途径分泌，这表明高阶寡聚体对于AtT-20细胞中的分选不是必需的。这些结果表明，当将该蛋白用作分泌途径中的报告分子时，必须考虑EGFP的寡聚化。
