We report here that importin α accumulates reversibly in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock. The nuclear accumulation of importin α appears to be triggered by a collapse in the Ran gradient, resulting in the suppression of the nuclear export of importin α. In addition, nuclear retention and the importin β/Ran-independent import of importin α also facilitate its rapid nuclear accumulation. The findings herein show that the classical nuclear import pathway is down-regulated via the removal of importin α from the cytoplasm in response to stress. Moreover, whereas the nuclear accumulation of heat shock cognate 70 is more sensitive to heat shock than the other stresses, importin α is able to accumulate in the nucleus at all the stress conditions tested. These findings suggest that the stress-induced nuclear accumulation of importin α can be involved in a common physiological response to various stress conditions.
Y.Miyamoto and M.Hieda contributed equally to this workA classical nuclear localization signal (NLS)-containing protein is transported into the nucleus via the formation of a NLS-substrate/importin a/b complex. In this study, we found that importin a migrated into the nucleus without the addition of importin b, Ran or any other soluble factors in an in vitro transport assay. A mutant importin a lacking the importin b-binding domain ef®ciently entered the nucleus. Competition experiments showed that this import pathway for importin a is distinct from that of importin b. These results indicate that importin a alone can enter the nucleus via a novel pathway in an importin b-and Ran-independent manner. Furthermore, this process is evolutionarily conserved as similar results were obtained in Saccharomyces cerevisiae. Moreover, the import rate of importin a differed among individual nuclei of permeabilized cells, as demonstrated by time-lapse experiments. This heterogeneous nuclear accumulation of importin a was affected by the addition of ATP, but not ATPgS. These results suggest that the nuclear import machinery for importin a at individual nuclear pore complexes may be regulated by reaction(s) that require ATP hydrolysis. Keywords: ATP/importin a/nuclear pore complex/ nuclear transport/time-lapse experiment IntroductionMolecular migration between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC) present in the double membrane of the nuclear envelope. The NPC, a huge proteinaceous structure composed of 50±100 different species of proteins called nucleoporins, is estimated to have a total mass of~125 MDa in higher eukaryotes and 66 MDa in Saccharomyces cerevisiae. Whereas molecules <20±40 kDa are able to passively diffuse through the NPC into the nucleus, the nuclear import of larger molecules is generally dependent on the presence of a speci®c signal sequence, the nuclear localization signal (NLS), and is often associated with a requirement for metabolic energy (Go Èrlich and Kutay, 1999). The ®rst identi®ed NLS was that of the SV40 large T-antigen, which consists of a short stretch of basic amino acids, designated as the basic type NLS. This type of NLS is divided into two groups, monopartite and bipartite, based on the number of basic amino acid clusters (Dingwall and Laskey, 1991).The nuclear import of basic type NLS-containing proteins is mediated by speci®c soluble factors that form a stable complex, the nuclear pore-targeting complex, in the cytoplasm (Imamoto et al., 1995a). The complex is composed of two essential components that are referred to as importin a and b (Go Èrlich and Mattaj, 1996). In addition to these molecules, several factors participate in this transport system, including a small GTPase Ran (Moore and Blobel, 1993) and Ran-binding proteins (Paschal and Gerace, 1995). Ran has a low intrinsic activity with respect to GDP/GTP exchange and GTP hydrolysis. RCC1, a guanine nucleotide exchange factor of Ran, accelerates the dissociation of the guanine nucleotide from Ran, ther...
In a previous study, we demonstrated that the forkhead associated (FHA) domain of pKi-67 interacts with the novel kinesin-like protein, Hklp2 (Sueishi, M., Takagi, M., and Yoneda, Y. (2000) J. Biol. Chem. 275, 28888 -28892). In this study, we report on the identification of a putative RNA-binding protein of 293 residues as another binding partner of the FHA domain of pKi-67 (referred to as NIFK for nucleolar protein interacting with the FHA domain of pKi-67). Human NIFK (hNIFK) interacted with the FHA domain of pKi-67 (Ki-FHA) efficiently in vitro when hNIFK was derived from mitotically arrested cells. In addition, a moiety of hNIFK was co-localized with pKi-67 at the peripheral region of mitotic chromosomes. The hNIFK domain that interacts with Ki-FHA was mapped in the yeast two-hybrid system to a portion encompassed by residues 226 -269. In a binding assay utilizing Xenopus egg extracts, it was found that the mitosis-specific environment and two threonine residues within this portion of hNIFK (Thr-234 and Thr-238) were crucial for the efficient interaction of hNIFK and Ki-FHA, suggesting that hNIFK interacts with Ki-FHA in a mitosis-specific and phosphorylation-dependent manner. These findings provide a new clue to our understanding of the cellular function of pKi-67.The Ki-67 antigen (pKi-67), originally identified as the antigen for a monoclonal antibody raised against the nuclear extract from a Hodgkin's lymphoma-derived cell line, was characterized as a class of proteins that localize around mitotic chromosomes (1). As a result, it is assumed that pKi-67 is involved in mitotic chromosome organization. pKi-67 is a convenient cell proliferation marker, since its expression is restricted to growing cells (2). Although the recent identification and characterization of a marsupial counterpart of pKi-67, which is referred to as chmadrin, suggests that pKi-67 plays some type of role in the organization of higher order chromatin structure (3), the actual role of pKi-67 in the cell cycle progression remains unclear.The N-terminal portion of pKi-67 is well conserved between human pKi-67 and chmadrin (62% identical) and contains a forkhead associated (FHA) 1 domain. It was originally reported that the FHA domain constituted a region that has been conserved in a subset of forkhead-type transcription factors (4). The sequence profile has been reported for a variety of proteins with diverse functions (transcription, DNA repair, cell cycle progression, etc.). In several instances, the FHA domain preferentially recognizes partner proteins when they are present in the phosphorylated form (5-8). Moreover, the strong specificity of the FHA domain for phosphopeptides has been clearly demonstrated by binding assays with synthetic phosphopeptides (9). Therefore, it is currently thought that the FHA domain is a general phosphopeptide recognition motif that is involved in certain phosphopeptide-mediated signal transduction pathways (10). A search for the interaction partner(s) of the FHA domain of pKi-67, which could exist in the...
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