The Rex protein of human T-cell leukemia virus type 1, like the functionally equivalent Rev protein of human immunodeficiency virus type 1, contains a leucine-rich activation domain that specifically interacts with the human nucleoporin-like Rab/hRIP cofactor. Here, this Rex sequence is shown to function also as a protein nuclear export signal (NES). Rex sequence libraries containing randomized forms of the activation domain/ NES were screened for retention of the ability to bind Rab/hRIP by using the yeast two-hybrid assay. While the selected sequences differed widely in primary sequence, all were functional as Rex activation domains. In contrast, randomized sequences that failed to bind Rab/hRIP lacked Rex activity. The selected sequences included one with homology to the Rev activation domain/NES and a second that was similar to the NES found in the cellular protein kinase inhibitor ␣. A highly variant, yet fully active, activation domain sequence selected on the basis of Rab/hRIP binding retained full NES function even though this sequence preserved only a single leucine residue. In contrast, nonfunctional activation domain mutants that were unable to bind Rab/hRIP had also lost NES function. These data demonstrate that NES activity is a defining characteristic of the activation domains found in the Rev/Rex class of retroviral regulatory proteins and strongly support the hypothesis that the Rab/hRIP cofactor plays a critical role in mediating the biological activity of these NESs. In addition, these data suggest a consensus sequence for NESs of the Rev/Rex class.The pathogenic complex retroviruses human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) belong to distinct retroviral families and display little sequence homology. The approaches used by these viruses to regulate proviral gene expression are nevertheless remarkably similar (reviewed in reference 5). Thus, each virus encodes a transcriptional regulatory protein that acts on the respective viral long terminal repeat promoter element to dramatically enhance viral gene expression. In addition, each virus also encodes an essential posttranscriptional regulatory protein, termed Rex in HTLV-1 and Rev in HIV-1, that induces the sequence-specific nuclear export, and hence translation, of the incompletely spliced viral mRNA species that encode the various viral structural proteins (8,11,16,17,20,23,27).Initial evidence favoring the hypothesis that Rex and Rev, despite lacking any significant sequence identity, might nevertheless mediate the nuclear export of target RNAs via the same mechanism came from the finding that Rex could partly rescue the replication of a Rev-deficient HIV-1 provirus (36). Subsequently, both Rev and Rex were shown to bind directly to structured RNA response elements present in their target RNAs (3,6,14,19,29,38,41) and to contain multimerization domains that are essential for the recruitment of additional Rev or Rex molecules (Fig. 1) (2, 26, 34). In addition, both Rev and Rex contain short, leu...
SummaryThe nefgene product encoded by the mac239 proviral done of simian immunodeficiency virus (SIV) markedly enhances viral replication and pathogenesis in vivo. We have used this biologicaUy active nefisolate to examine the phenotype of Nef in retrovirally transduced human T cells in culture. SIV Nefis shown to dramatically inhibit cell-surface expression of the CD4 glycoprorein without significantly affecting the total steady-state level of cellular CD4. This downregulation of the cell-surface CD4 receptor for human immunodeficiency virus type 1 (HIV-1) infection correlated with the acquisition of resistance to superinfection by HIV-1. However, SIV Nef did not affect the level of gene expression directed by the HIV-1 long terminal repeat. It is hypothesized that downregulation of cell-surface CD4 by Nef facilitates the efficient release of infectious progeny virions and, hence, viral spread in vivo.
The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin ␣/importin ␣, which acts as the NLS receptor, and karyopherin 1/importin , which binds karyopherin ␣ and mediates the nuclear import of the resultant ternary complex. Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified. In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described. The M9 NLS binds a transport factor related to karyopherin 1, termed karyopherin 2 or transportin, and does not require a karyopherin ␣-like adapter protein. A yeast homolog of karyopherin 2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p. Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs. Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS. In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p. Surprisingly, this in vitro assay also revealed that human karyopherin 1, but not the Kap104p homolog karyopherin 2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin ␣. These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences.The regulated movement of macromolecules into and out of the nucleus is essential for the viability of eukaryotic cells, and several distinct nuclear import and export pathways are believed to exist (reviewed in references 17 and 40). Of these, the best understood is the pathway that mediates the nuclear import of proteins bearing basic nuclear localization signals (NLSs) of the type first described for simian virus 40 (SV40) T antigen and nucleoplasmin (12,26). Import of such proteins is initiated by the direct interaction of the basic NLS with karyopherin ␣ (also termed importin ␣) (2,19,24,35,37,50), which in turn is bound by a second component of the nuclear import machinery termed karyopherin 1 (also termed importin ) (9, 20, 35, 37). The resultant heterotrimer is then recruited to the cytoplasmic face of a nuclear pore by the direct interaction of karyopherin 1 with nucleoporins (35, 37, 43). The subsequent transition of this heterotrimeric receptor complex into the nucleus is mediated by the cellular Ran GTPase (27, 32, 34) and by a second cofactor termed p10 or NTF2 (10, 39, 41). Directionality of movement is thought to result from the ordered, sequential interaction of the karyopherin 1 subunit with specific nucleoporins (44). Protein i...
Fragile X syndrome results from lack of expression of a functional form of Fragile X mental retardation protein (FMRP), a cytoplasmic RNA‐binding protein of uncertain function. Here, we report that FMRP contains a nuclear export signal (NES) that is similar to the NES recently identified in the Rev regulatory protein of human immunodeficiency virus type 1 (HIV‐1). Mutation of this FMRP NES results in mis‐localization of FMRP to the cell nucleus. The FMRP NES is encoded within exon 14 of the FMR1 gene, thus explaining the aberrant nuclear localization of a natural isoform of FMRP that lacks this exon. The NES of FMRP can substitute fully for the Rev NES in mediating Rev‐dependent nuclear RNA export and specifically binds a nucleoporin‐like cellular cofactor that has been shown to mediate Rev NES function. Together, these findings demonstrate that the normal function of FMRP involves entry into the nucleus followed by export via a pathway that is identical to the one utilized by HIV‐1 Rev. In addition, these data raise the possibility that FMRP could play a role in mediating the nuclear export of its currently undefined cellular RNA target(s).
The low cytoplasmic and high nuclear concentration of the GTP-bound form of Ran provides directionality for both nuclear protein import and export. Both import and export factors bind RanGTP directly, yet this interaction produces opposite effects; in the former case, RanGTP binding induces nuclear cargo release, whereas in the latter, RanGTP binding induces nuclear cargo assembly. Therefore, nuclear import and export receptors and their protein recognition sites are predicted to be distinct. Nevertheless, the ϳ38-amino acid M9 sequence present in heterogeneous nuclear ribonucleoprotein A1 has been reported to serve as both a nuclear localization signal and a nuclear export signal, even though only one protein, the nuclear import factor transportin, has been shown to bind M9 directly. We have used a combination of mutational randomization followed by selection for transportin binding to exhaustively define amino acids in M9 that are critical for transportin binding in vivo. As expected, the resultant ϳ12-amino acid transportin-binding consensus sequence is also predictive of nuclear localization signal activity. Surprisingly, however, this extensive mutational analysis failed to dissect M9 nuclear localization signal and nuclear export signal function. Nevertheless, transportin appears unlikely to be the M9 export receptor, as RanGTP can be shown to block M9 binding by transportin not only in vitro, but also in the nucleus in vivo. This analysis therefore predicts the existence of a nuclear export receptor distinct from transportin that nevertheless shares a common protein-binding site on heterogeneous nuclear ribonucleoprotein A1.Recent progress in understanding the mechanisms that drive the nucleocytoplasmic transport of proteins and RNAs suggests that both nuclear import and export are mediated by a family of proteins related to the prototypic import factor importin /karyopherin 1 (Imp-) 1 (reviewed in Refs. 1-3). Directionality of movement appears, at least in part, to be controlled by the low cytoplasmic and high nuclear concentration of the GTP-bound form of Ran (4, 5). Thus, import factors, such as Imp-, are believed to bind to nuclear localization signals (NLSs) or adaptor molecules, such as importin/karyopherin ␣, in the cytoplasm, where RanGTP is found at very low concentrations. Once the resultant import receptor-substrate complex reaches the nucleus, where RanGTP exists at high concentrations, the direct interaction of Imp- with RanGTP induces the release of the protein cargo (5-7). Conversely, Imp--related nuclear export factors such as Cas and Crm1 are believed to bind proteins containing a cognate nuclear export signal (NES) in the nucleus only in the form of a ternary complex involving RanGTP (8, 9). Once the resultant export receptor-substrate complex reaches the cytoplasm, RanGTP is hydrolyzed to RanGDP by the cytoplasmic RanGAP and RanBP1 or RanBP2 proteins, thereby inducing the release of the export receptor from both Ran and the export substrate.A prediction of the hypothesis that RanG...
We describe a "protein knockout" technique that can be used to identify essential proteins in bacteria. This technique uses phage display to select peptides that bind specifically to purified target proteins. The peptides are expressed intracellularly and cause inhibition of growth when the protein is essential. In this study, peptides that each specifically bind to one of seven essential proteins were identified by phage display and then expressed as fusions to glutathione S-transferase in Escherichia coli. Expression of peptide fusions directed against E. coli DnaN, LpxA, RpoD, ProRS, SecA, GyrA, and Era each dramatically inhibited cell growth. Under the same conditions, a fusion with a randomized peptide sequence did not inhibit cell growth. In growthinhibited cells, inhibition could be relieved by concurrent overexpression of the relevant target protein but not by coexpression of an irrelevant protein, indicating that growth inhibition was due to a specific interaction of the expressed peptide with its target. The protein knockout technique can be used to assess the essentiality of genes of unknown function emerging from the sequencing of microbial genomes. This technique can also be used to validate proteins as drug targets, and their corresponding peptides as screening tools, for discovery of new antimicrobial agents.
Heterogeneous nuclear ribonucleoprotein A1 contains a sequence, termed M9, that functions as a potent nuclear localization signal (NLS) yet bears no similarity to the well-defined basic class of NLSs. Here, we report the identification of a novel human protein, termed MIP, that binds M9 specifically both in vivo and in vitro yet fails to interact with non-functional M9 point mutants. Of note, the 101 kDa MIP protein bears significant homology to human karyopherin/importin-beta, a protein known to mediate the function of basic NLSs. The in vitro nuclear import of a protein substrate containing the M9 NLS was found to be dependent on provision of the MIP protein in trans. Cytoplasmic microinjection of a truncated form of MIP that retains the M9 binding site blocked the in vivo nuclear import of a substrate containing the M9 NLS yet failed to affect the import of a similar substrate bearing a basic NLS. These data indicate that nuclear import of hnRNP A1 is mediated by a novel cellular import pathway that is distinct from, yet evolutionarily related to, the pathway utilized by basic NLS sequences.
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