Tang Y, DeFranco D B
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.
Mol Cell Biol. 1996 May;16(5):1989-2001. doi: 10.1128/MCB.16.5.1989.
Glucocorticoid receptors (GRs) have the capacity to shuttle between the nuclear and cytoplasmic compartments, sharing that trait with other steroid receptors and unrelated nuclear proteins of diverse function. Although nuclear import of steroid receptors, like that of nearly all other karyophilic proteins examined to date, requires ATP, there appear to be different energetic requirements for export of proteins, including steroid receptors, from nuclei. In an attempt to reveal which steps, if any, in the nuclear export pathway utilized by steroid receptors require ATP, we have used indirect immunofluorescence to visualize GRs within cells subjected to a reversible ATP depletion. Under conditions which lead to >95% depletion of cellular ATP levels within 90 min, GRs remain localized within nuclei and do not efflux into the cytoplasm. Under analogous conditions of ATP depletion, transfected progesterone receptors are also retained within nuclei. Importantly, GRs which accumulate within nuclei of ATP-depleted cells are distinguished from nuclear receptors in metabolically active cells by their resistance to in situ extraction with a hypotonic, detergent-containing buffer. GRs in ATP-depleted cells are not permanently trapped in this nuclear compartment, as nuclear receptors rapidly regain their capacity to be extracted upon restoration of cellular ATP, even in the absence of de novo protein synthesis. More extensive extraction of cells with high salt and detergent, coupled with DNase I digestion, established that a significant fraction of GRs in ATP-depleted cells are associated with an RNA-containing nuclear matrix. Quantitative Western blot (immunoblot) analysis confirmed the dramatic increase in GR binding to the nuclear matrix of ATP-depleted cells, while confocal microscopy revealed that GRs are bound to the matrix throughout all planes of the nucleus. ATP depletion does not lead to wholesale collapse of nuclear proteins onto the matrix, as the interaction of a subpopulation of simian virus 40 large tumor antigen with the nuclear matrix is not quantitatively altered in ATP-depleted Cos-1 cells. Nuclear GRs which are not bound to the nuclear matrix of metabolically active cells (i.e., a DNA-binding domain deletion mutant and a beta-galactosidase chimera possessing the GR nuclear localization signal sequence) are not recruited to the matrix upon depletion of cellular ATP. Thus, it appears that ATP depletion does not expose the GR to nuclear matrix interactions which are not normally encountered in cells but merely alters the dynamics of such interactions. The dynamic association of steroid receptors with the nuclear matrix may provide a mechanism which is utilized by these regulable transcription factors to facilitate their efficient scanning of the genome.
糖皮质激素受体（GRs）能够在细胞核和细胞质区室之间穿梭，这一特性与其他类固醇受体以及功能多样的非相关核蛋白相同。尽管类固醇受体的核输入，如同迄今为止检测的几乎所有其他亲核蛋白一样，需要ATP，但蛋白质（包括类固醇受体）从细胞核输出似乎有不同的能量需求。为了揭示类固醇受体利用的核输出途径中哪些步骤（如果有的话）需要ATP，我们利用间接免疫荧光来观察处于可逆性ATP耗竭状态的细胞内的GRs。在90分钟内导致细胞ATP水平耗竭>95%的条件下，GRs仍定位于细胞核内，不会外流到细胞质中。在类似的ATP耗竭条件下，转染的孕激素受体也保留在细胞核内。重要的是，在ATP耗竭细胞的细胞核内积累的GRs与代谢活跃细胞中的核受体不同，它们对用含有去污剂的低渗缓冲液进行原位提取具有抗性。ATP耗竭细胞中的GRs不会永久被困在这个核区室中，因为即使在没有从头合成蛋白质的情况下，细胞ATP恢复后，核受体也会迅速恢复其被提取的能力。用高盐和去污剂对细胞进行更广泛的提取，并结合DNase I消化，证实ATP耗竭细胞中相当一部分GRs与含RNA的核基质相关。定量蛋白质免疫印迹分析证实了ATP耗竭细胞中GR与核基质结合的显著增加，而共聚焦显微镜显示GRs在细胞核的所有平面上都与基质结合。ATP耗竭不会导致核蛋白整体坍塌到基质上，因为在ATP耗竭的Cos-1细胞中，猿猴病毒40大肿瘤抗原亚群与核基质的相互作用在数量上没有改变。在代谢活跃细胞中不与核基质结合的核GRs（即DNA结合结构域缺失突变体和具有GR核定位信号序列的β-半乳糖苷酶嵌合体）在细胞ATP耗竭时不会被招募到基质上。因此，似乎ATP耗竭并没有使GR暴露于细胞中通常不会遇到的核基质相互作用，而只是改变了这种相互作用的动力学。类固醇受体与核基质的动态结合可能提供了一种机制，这些可调节的转录因子利用该机制来促进它们对基因组的有效扫描。
