Showing papers on "Importin published in 1994"

RanGAP1 induces GTPase activity of nuclear Ras-related Ran.

Abstract: The nuclear Ras-related protein Ran binds guanine nucleotide and is involved in cell cycle regulation. Models of the signal pathway predict Ran to be active as Ran.GTP at the initiation of S phase upon activation by the nucleotide exchange factor RCC1 and to be inactivated for the onset of mitosis by hydrolysis of bound GTP. Here a nuclear homodimeric 65-kDa protein, RanGAP1, is described, which we believe to be the immediate antagonist of RCC1. It was purified from HeLa cell lysates and induces GTPase activity of Ran, but not Ras, by more than 3 orders of magnitude. The Ran mutant Q69L, modeled after RasQ61L, which is unable to hydrolyze bound GTP, is insensitive to RanGAP1.

Nuclear localization of p85s6k: functional requirement for entry into S phase.

Abstract: Immunolocalization of a newly described isoform of p70s6k, termed p85s6k, demonstrated a predominantly nuclear location in rat embryo fibroblasts (REF-52), a compartment in which growth factor-mediated phosphorylation of S6 has recently been reported. Microinjection of expression vectors encoding either p85s6k or a fusion protein containing only the putative nuclear localization motifs led to the exclusive accumulation of both products in the nucleus. Consistent with such a localization, microinjection of affinity-purified anti-p85s6k IgG into the nucleus, but not the cytoplasm, blocked serum-induced initiation of DNA synthesis. Co-injection into the nucleus of the anti-p85s6k IgG with activated p70s6k, which lacks the antigenic epitope, rescued the S phase block, arguing that the antibody exerts its effects through inhibiting p85s6k function. The results indicate a novel role for S6 phosphorylation in the nucleus distinct from that in the cytoplasm, a role essential for mitogenesis.

A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm.

Abstract: RNA-binding proteins have been suggested to move in association with RNA as it leaves the nucleus. The NPL3 gene of the yeast Saccharomyces cerevisiae encodes in nuclear protein with consensus RNA-binding motifs and similarity to heterogeneous nuclear ribonucleoproteins and members of the S/R protein family. We show that although Npl3 is located in the nucleus, it can shuttle between nuclei in yeast heterokaryons. In contrast, other nucleus-targeted proteins do not leave the nucleus under similar conditions. Mutants missing the RNA-binding motifs or the N terminus are still capable of shuttling in and out of the nucleus. Npl3 mutants missing the C terminus fail to localize to the nucleus. Overproduction of Npl3 in wild-type cells shows cell growth. This toxicity depends on the presence of series of unique repeats in the N terminus and localization to the nucleus. We suggest that the properties of Npl3 are consistent with it being involved in export of RNAs from the nucleus.

A mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a regulator of chromosome condensation.

Abstract: The Ran protein is a small GTPase that has been implicated in a large number of nuclear processes including transport. RNA processing and cell cycle checkpoint control. A similar spectrum of nuclear activities has been shown to require RCC1, the guanine nucleotide exchange factor (GEF) for Ran. We have used the Xenopus laevis egg extract system and in vitro assays of purified proteins to examine how Ran or RCC1 could be involved in these numerous processes. In these studies, we employed mutant Ran proteins to perturb nuclear assembly and function. The addition of a bacterially expressed mutant form of Ran (T24N-Ran), which was predicted to be primarily in the GDP-bound state, profoundly disrupted nuclear assembly and DNA replication in extracts. We further examined the molecular mechanism by which T24N-Ran disrupts normal nuclear activity and found that T24N-Ran binds tightly to the RCC1 protein within the extract, resulting in its inactivation as a GEF. The capacity of T24N-Ran-blocked interphase extracts to assemble nuclei from de-membranated sperm chromatin and to replicate their DNA could be restored by supplementing the extract with excess RCC1 and thereby providing excess GEF activity. Conversely, nuclear assembly and DNA replication were both rescued in extracts lacking RCC1 by the addition of high levels of wild-type GTP-bound Ran protein, indicating that RCC1 does not have an essential function beyond its role as a GEF in interphase Xenopus extracts.

Nuclear localization signals also mediate the outward movement of proteins from the nucleus

Abstract: Several nuclear proteins, including steroid hormone receptors, have been shown to shuttle continuously between the nucleus and the cytoplasm. The mechanism of entry of proteins into the nucleus is well documented, whereas the mechanism of their outward movement into the cytoplasm is not understood. We have grafted the nuclear localization signals of the progesterone receptor or the simian virus 40 large tumor antigen onto beta-galactosidase. These additions were shown to impart to the protein the ability to shuttle between the nucleus and the cytoplasm. Microinjected proteins devoid of a nuclear localization signal were unable to exit from the nucleus. The same nuclear localization signals are thus involved in both the inward and the outward movement of proteins through the nuclear membrane. We also show that although the nuclear import requires energy, the nuclear export does not. These results suggest that the nucleocytoplasmic shuttling may be a general phenomenon for nuclear proteins that could possibly undergo modifications in the cytoplasm and exert some biological activities there. These conclusions also imply that at least part of the cellular machinery involved in the nuclear import of proteins may function bidirectionally.

Effects of mutant Ran/TC4 proteins on cell cycle progression.

Abstract: Ran/TC4, a member of the RAS gene superfamily, encodes an abundant nuclear protein that binds and hydrolyzes GTP. Transient expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis blocked DNA replication, suggesting a role for Ran/TC4 in the regulation of cell cycle progression. To test this possibility, we exploited an efficient transfection system, involving the introduction of cDNAs in the pMT2 vector into 293/Tag cells, to analyze phenotypes associated with mutant and wild-type Ran/TC4 expression. Expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis inhibited proliferation of transfected cells by arresting them predominantly in the G2, but also in the G1, phase of the cell cycle. Deletion of an acidic carboxy-terminal hexapeptide from the Ran/TC4 mutant did not alter its nuclear localization but did block its inhibitory effect on cell cycle progression. These data suggest that normal progression of the cell cycle is coupled to the operation of a Ran/TC4 GTPase cycle. Mediators of this coupling are likely to include the nuclear regulator of chromosome condensation 1 protein and the mitosis-promoting factor complex.

The U1 small nuclear ribonucleoprotein (snRNP) 70K protein is transported independently of U1 snRNP particles via a nuclear localization signal in the RNA-binding domain.

Abstract: Expression of the recombinant human U1-70K protein in COS cells resulted in its rapid transport to the nucleus, even when binding to U1 RNA was debilitated. Deletion analysis of the U1-70K protein revealed the existence of two segments of the protein which were independently capable of nuclear localization. One nuclear localization signal (NLS) was mapped within the U1 RNA-binding domain and consists of two typically separated but interdependent elements. The major element of this NLS resides in structural loop 5 between the beta 4 strand and the alpha 2 helix of the folded RNA recognition motif. The C-terminal half of the U1-70K protein which was capable of nuclear entry contains two arginine-rich regions, which suggests the existence of a second NLS. Site-directed mutagenesis of the RNA recognition motif NLS demonstrated that the U1-70K protein can be transported independently of U1 RNA and that its association with the U1 small nuclear ribonucleoprotein particle can occur in the nucleus.

Genetically defined nuclear localization signal sequence of bovine papillomavirus E1 protein is necessary and sufficient for the nuclear localization of E1-beta-galactosidase fusion proteins.

Abstract: The 605 amino acid E1 protein of bovine papillomavirus type 1 (BPV-1) is a multifunctional nuclear protein required for viral DNA replication. A nuclear localization signal (NLS) sequence was previously defined by point mutations in three short adjacent clusters of basic amino acids located in the amino-terminal region of the E1 protein. In this study, we used a fusion protein approach to evaluate the contribution of other regions of the E1 protein to nuclear transport. The nearly full-length E1 gene and six non-overlapping subfragments were each fused in-frame with the lacZ gene in a eukaryotic expression vector. Each clone was electro-porated into COS-1 cells, and the intracellular location of the E1-β-galactosidase fusion proteins was determined by immunofluorescence. Only the constructs containing the full-length E1 or a single subregion (E1-259; amino acids 84 to 166) produced fusion proteins that entered the nucleus. Point mutations in the NLS sequences of the E1-259-lacZ construct prevented nuclear translocation of the corresponding fusion protein. This confirms the previous result that the cluster of basic amino acids is critical for nuclear transport. Furthermore, the data obtained in this investigation indicated that the region of E1 containing the NLS sequence was not only necessary, but was also sufficient for nuclear localization. No other region of E1 contained independent nuclear localization activity.