Molecular chaperones function as steroid receptor nuclear mobility factors

Abstract

Live cell imaging has revealed the rapid mobility of steroid hormone receptors within nuclei and their dynamic exchange at transcriptionally active target sites. Although a number of other proteins have been shown to be highly mobile within nuclei, the identity of soluble factors responsible for orchestrating nuclear trafficking remains unknown. We have developed a previously undescribed in situ subnuclear trafficking assay that generates transcriptionally active nuclei, which are depleted of soluble factors required for the nuclear mobility of glucocorticoid (GR) and progesterone receptors (PR). Using this system and a fluorescence recovery after photobleaching technique, we demonstrate that nuclear mobility of GR recovered on incubation with reticulocyte lysate was inhibited by geldanamycin, a drug that blocks the chaperone activity of heat-shock protein 90. Direct proof of molecular chaperone involvement in steroid receptor subnuclear trafficking was provided by the ATP-dependent recovery of nuclear mobility of GR and PR on incubation with various combinations of purified chaperone and͞or cochaperone proteins. Additionally, for both receptors, the inclusion of hormone during the recovery period leads to a retardation of nuclear mobility. Thus, our results provide a description of soluble nuclear mobility factors and furthermore demonstrate a previously unrecognized role for molecular chaperones in the regulation of steroid receptor function within the nucleus.O ur understanding of the mechanisms of protein trafficking has been greatly aided by the advent of technologies that allow the movement of individual proteins to be visualized in live cells (1, 2). When applied to the assessment of nuclear protein trafficking, such techniques have uniquely revealed details of protein movement within a single subcellular compartment (3-6). Although protein mobility within the nucleus can be diffusion-limited, low-or high-affinity interactions with specific soluble or solid-state targets can reduce the kinetics of nuclear protein trafficking (7). Nonetheless, nuclear proteins that are components of large macromolecular complexes are highly mobile and possess fast diffusion coefficients (7).The nuclear receptor superfamily of proteins act as transcription factors with activities regulated by ligand binding (8,9). Once associated with their target genes through direct interactions with specific DNA sequences or tethering to other DNA-bound factors, nuclear receptors recruit large coregulator complexes to linked promoters (10, 11). These coregulators include coactivator and corepressor complexes that modulate RNA polymerase II activity in part through direct modification of histone proteins within core nucleosomes (12)(13)(14). The interaction of specific components (e.g., SRC-1) of coactivator complexes with a ligand-bound nuclear receptor (e.g., estrogen receptor) reduces their mobility within live cell nuclei, perhaps reflecting the assembly of functional coactivator͞nuclear receptor complexes on active target genes (15)....

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Chromatin Remodeling Complexes Interact Dynamically with a Glucocorticoid Receptor–regulated Promoter

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Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle

“…The rapid in vivo exchange rates appear to be a result of several factors, including: the active displacement of proteins by another copy of the same protein, the presence of chromatin remodeling complexes that actively displace proteins, ATP levels and post-translation modifications [60][61][62][63][64][65][66][67][68][69]. In addition, decreasing the temperature at which the cells are photobleached results in reduced protein mobility [60,66,70]. In biochemical applications, cells are permeabilized at low temperatures, which would result in both the dilution of soluble factors and a decrease in mobility, giving the impression of a statically bound protein.…”