Wooden S K, Kapur R P, Lee A S
Department of Biochemistry, University of Southern California, School of Medicine, Los Angeles 90033.
Exp Cell Res. 1988 Sep;178(1):84-92. doi: 10.1016/0014-4827(88)90380-1.
Expression of the glucose-regulated protein, GRP78, is markedly increased when cells are placed in a variety of stressful environments (i.e., low glucose medium, calcium ionophore treatment). In this report, the genomic organization of the rat GRP78 gene is described. This gene comprises eight exons and encodes a protein which is highly hydrophilic with the notable exception of several short hydrophobic domains. The first hydrophobic region, 18 amino acids at the N-terminus of the protein, putatively acts as a signal sequence to target GRP78 into the endoplasmic reticulum (ER). By ligating portions of the GRP78 gene and its promoter to the bacterial gene encoding chloramphenicol acetyltransferase (CAT), we created heterologous CAT genes inducible by calcium ionophore A23187. Through immunofluorescence analysis, the intracellular localizations of endogenous GRP78 and fusion CAT proteins under normal growth and A23187-induced conditions are identified. By fusing the GRP78 signal sequence to CAT, we influence intracellular targeting of the CAT protein into the ER.
当细胞置于各种应激环境（如低糖培养基、钙离子载体处理）中时，葡萄糖调节蛋白GRP78的表达会显著增加。在本报告中，描述了大鼠GRP78基因的基因组结构。该基因由八个外显子组成，编码一种高度亲水的蛋白质，但有几个短的疏水结构域除外。第一个疏水区域位于蛋白质的N端，由18个氨基酸组成，推测作为信号序列将GRP78靶向内质网（ER）。通过将GRP78基因及其启动子的部分片段与编码氯霉素乙酰转移酶（CAT）的细菌基因连接，我们创建了可被钙离子载体A23187诱导的异源CAT基因。通过免疫荧光分析，确定了正常生长和A23187诱导条件下内源性GRP78和融合CAT蛋白的细胞内定位。通过将GRP78信号序列与CAT融合，我们影响了CAT蛋白进入内质网的细胞内靶向。
