Mercier P A, Winegarden N A, Westwood J T
Department of Zoology, University of Toronto, Mississauga, Ontario, Canada L5L 1C6.
J Cell Sci. 1999 Aug;112 ( Pt 16):2765-74. doi: 10.1242/jcs.112.16.2765.
The induction of the heat shock genes in eukaryotes by heat and other forms of stress is mediated by a transcription factor known as heat shock factor 1 (HSF1). HSF1 is present in unstressed metazoan cells as a monomer with low affinity for DNA, and upon exposure to stress it is converted to an 'active' homotrimer that binds the promoters of heat shock genes with high affinity and induces their transcription. The conversion of HSF1 to its active form is hypothesized to be a multistep process involving physical changes in the HSF1 molecule and the possible translocation of HSF1 from the cytoplasm to the nucleus. While all studies to date have found active HSF1 to be a nuclear protein, there have been conflicting reports on whether the inactive form of HSF is predominantly a cytoplasmic or nuclear protein. In this study, we have made antibodies against human HSF1 and have reexamined its localization in unstressed and heat-shocked human HeLa and A549 cells, and in green monkey Vero cells. Biochemical fractionation of heat-shocked HeLa cells followed by western blot analysis showed that HSF1 was mostly found in the nuclear fraction. In extracts made from unshocked cells, HSF1 was predominantly found in the cytoplasmic fraction using one fractionation procedure, but was distributed approximately equally between the cytoplasmic and nuclear fractions when a different procedure was used. Immunofluorescence microscopy revealed that HSF1 was predominantly a nuclear protein in both heat shocked and unstressed cells. Quantification of HSF1 staining showed that approximately 80% of HSF1 was present in the nucleus both before and after heat stress. These results suggest that HSF1 is predominantly a nuclear protein prior to being exposed to stress, but has low affinity for the nucleus and is easily extracted using most biochemical fractionation procedures. These results also imply that HSF1 translocation is probably not part of the multistep process in HSF1 activation for many cell types.
热和其他形式的应激在真核生物中诱导热休克基因是由一种称为热休克因子1（HSF1）的转录因子介导的。HSF1在未受应激的后生动物细胞中以对DNA亲和力低的单体形式存在，在受到应激时，它会转变为“活性”同源三聚体，该三聚体以高亲和力结合热休克基因的启动子并诱导其转录。HSF1向其活性形式的转变被认为是一个多步骤过程，涉及HSF1分子的物理变化以及HSF1可能从细胞质转移到细胞核。虽然迄今为止所有研究都发现活性HSF1是一种核蛋白，但关于HSF的非活性形式主要是细胞质蛋白还是核蛋白一直存在相互矛盾的报道。在本研究中，我们制备了针对人HSF1的抗体，并重新检查了其在未受应激和热休克的人HeLa和A549细胞以及绿猴Vero细胞中的定位。对热休克的HeLa细胞进行生化分级分离，然后进行蛋白质印迹分析，结果表明HSF1主要存在于细胞核分级分离物中。在未受冲击的细胞提取物中，使用一种分级分离方法时，HSF1主要存在于细胞质分级分离物中，但使用不同方法时，它在细胞质和细胞核分级分离物之间的分布大致相等。免疫荧光显微镜检查显示，在热休克和未受应激的细胞中，HSF1主要是一种核蛋白。HSF1染色的定量分析表明，热应激前后约80%的HSF1存在于细胞核中。这些结果表明，HSF1在受到应激之前主要是一种核蛋白，但对细胞核的亲和力较低，并且使用大多数生化分级分离程序很容易提取。这些结果还意味着，对于许多细胞类型，HSF1易位可能不是HSF1激活多步骤过程的一部分。
