Mori K, Sant A, Kohno K, Normington K, Gething M J, Sambrook J F
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038.
EMBO J. 1992 Jul;11(7):2583-93. doi: 10.1002/j.1460-2075.1992.tb05323.x.
The KAR2 gene of Saccharomyces cerevisiae codes for an essential chaperone protein (BiP) that is localized in the lumen of the endoplasmic reticulum (ER). The high basal rate of transcription of KAR2 is increased transiently by heat shock: prolonged induction occurs when unfolded proteins accumulate in the ER. Three cis-acting elements in the KAR2 promoter control expression of KAR2: (i) a GC-rich region that contributes to the high level of constitutive expression, (ii) a functional heat shock element (HSE) and (iii) an element (UPR) that is involved in the induction of BiP mRNA by unfolded proteins. By analyzing internal deletion mutants of the KAR2 promoter, we demonstrate here that these three elements regulate transcription of KAR2 independently. Furthermore, the 22 bp UPR element causes a heterologous (CYC1) promoter to respond to the presence of unfolded proteins in the ER. Extracts of both stressed and unstressed yeast cells contain proteins that bind specifically to synthetic HSE and UPR elements and retard their migration through gels. Binding proteins specific for the UPR element can be fractionated by ammonium sulfate precipitation. Two of the proteins UPRF-1 and UPRF-2 (which is apparently a proteolytic degradation product of UPRF-1) bind inefficiently to mutant versions of the UPR that are unable to confer responsiveness to unfolded proteins to the (CYC1) promoter. UPRF-1 therefore displays the properties expected of a transcription factor that is involved in the sustained response of the KAR2 promoter to unfolded proteins in the ER. These experiments show that yeast cells can activate a transcription factor that stimulates expression of a nuclear gene in response to the accumulation of unfolded proteins in another cellular compartment.
酿酒酵母的KAR2基因编码一种内质网（ER）腔中定位的必需伴侣蛋白（BiP）。KAR2的高基础转录率在热休克时会短暂增加：当未折叠蛋白在内质网中积累时会发生长时间诱导。KAR2启动子中的三个顺式作用元件控制KAR2的表达：（i）一个富含GC的区域，有助于高水平的组成型表达；（ii）一个功能性热休克元件（HSE）；（iii）一个参与未折叠蛋白诱导BiP mRNA的元件（UPR）。通过分析KAR2启动子的内部缺失突变体，我们在此证明这三个元件独立调节KAR2的转录。此外，22 bp的UPR元件使异源（CYC1）启动子对内质网中未折叠蛋白的存在作出反应。应激和未应激酵母细胞的提取物都含有能特异性结合合成HSE和UPR元件并使其在凝胶中迁移受阻的蛋白质。UPR元件特异性结合蛋白可以通过硫酸铵沉淀进行分级分离。其中两种蛋白UPRF-1和UPRF-2（显然是UPRF-1的蛋白水解降解产物）与无法赋予（CYC1）启动子对未折叠蛋白反应性的UPR突变体结合效率低下。因此，UPRF-1显示出参与KAR2启动子对内质网中未折叠蛋白持续反应的转录因子的预期特性。这些实验表明，酵母细胞可以激活一种转录因子，该转录因子能响应另一个细胞区室中未折叠蛋白的积累来刺激核基因的表达。
