Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells
Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells
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DOI：
10.1042/0264-6021:3600645
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发表时间：
2001-12-15
影响因子：
4.1
通讯作者：
Gorr, SU
中科院分区：
文献类型：
--
作者：
Jain, RK;Joyce, PBM;Gorr, SU
绿色荧光蛋白（GFP）被广泛用作报告蛋白来监测细胞过程，包括细胞内蛋白质运输和分泌。通常，这种方法依赖于GFP作为被动报告蛋白，然而，最近注意到GFP在内分泌细胞的分泌途径中寡聚化。为了表征这种寡聚化及其在GFP转运中的潜在作用，在GH4 C1和AtT-20内分泌细胞中表达胞质和分泌形式的增强型GFP（EGFP）。生化分析表明，细胞溶质的EGFP存在作为一个27 kDa的单体，而分泌形式的EGFP形成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）），这可能在这种寡聚化中起作用。Cys（49）和Cys（71）的定点突变显示两个半胱氨酸残基都参与二硫键相互作用。取代半胱氨酸残基导致减少或损失的低聚物，虽然二聚体的分泌形式的EGFP仍然存在。这些残基的突变没有不利地影响EGFP的荧光。增强型绿色荧光蛋白寡聚体储存在分泌颗粒中，并通过内分泌AtT-20细胞的调节分泌途径分泌。类似地，EGFP的二聚体突变体形式仍然通过受调节的分泌途径分泌，这表明高级寡聚体对于AtT-20细胞中的分选是不必要的。这些结果表明，EGFP的寡聚化，必须考虑当蛋白质被用作报告分子在分泌途径。
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.