Reconstitution of HIV-1 Rev nuclear export: Independent requirements for nuclear import and export

Abstract

The Rev protein of HIV-1 actively shuttles between nucleus and cytoplasm and mediates the export of unspliced retroviral RNAs. The localization of shuttling proteins such as Rev is controlled by the relative rates of nuclear import and export. To study nuclear export in isolation, we generated cell lines expressing a green f luorescent proteinlabeled chimeric protein consisting of HIV-1 Rev and a hormone-inducible nuclear localization sequence. Steroid removal switches off import thus allowing direct visualization of the Rev export pathway in living cells. After digitonin permeabilization of these cells, we found that a functional nuclear export sequence (NES), ATP, and fractionated cytosol were sufficient for nuclear export in vitro. Nuclear pore-specific lectins and leptomycin B were potent export inhibitors. Nuclear export was not inhibited by antagonists of calcium metabolism that block nuclear import. These data further suggest that nuclear pores do not functionally close when luminal calcium stores are depleted. The distinct requirements for nuclear import and export argue that these competing processes may be regulated independently. This system should have wide applicability for the analysis of nuclear import and export.The bidirectional exchange of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (1). Generally, the cut-off between facilitated transport and diffusion across the nuclear pore complex is 40-50 kDa. However, some smaller molecules such as histone H1 also are transported actively (2). Active transport is a highly regulated process requiring specific targeting sequences, receptors, regulatory proteins, and energy (reviewed in refs. 3 and 4). Nuclear import also requires the small GTPase Ran, p10, and GTP hydrolysis (5, 6). In addition, alternate pathways for nuclear import exist (7,8). Although many of the requirements of import have been identified, the exact mechanism of translocation across the pore is poorly understood.Nuclear export, like import, is also initiated by a specific sequence. One of the first nuclear export sequences (NESs) was identified in the HIV-1 transactivating protein Rev (9). This hydrophobic sequence, LPPLERLTLD, is characterized by critical leucine residues; removal of one of these residues inactivates this sequence and prevents export (10). Similar NESs have been identified in several other shuttling proteins (11)(12)(13)(14)(15)(16)(17)(18)(19). When the NES is placed on a heterologous protein, it is sufficient to initiate export. An excess amount of the Rev NES peptide competes efficiently with some RNA species including 5S rRNA and spliceosomal U snRNAs for export out of the nucleus (9, 20). These competition studies indicate that Rev utilizes an established nuclear export pathway within the cell.NES-containing proteins are exported in an active and saturable manner, indicating that the process is receptor-mediated. However, identification of export receptors has proven more difficult than the isolation of import r...

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“…3, ϩHϩ2h chase, and Ref. 63), although the Rev NES is able to induce a complete nuclear export in the same experimental condition (67). In contrast, hormone removal did not significantly affect the subcellular distribution of GR nor IB␣ 37-55-GR (Fig.…”

“…When the HIV-1 Rev protein was fused to the hormone-binding region of GR, the chimeric protein was completely imported into the nucleus and nucleolus upon hormone treatment, indicating that fusion of a very efficient NES to GR is not sufficient to delocalize hormone-bound GR to the cytoplasm. In contrast, hormone withdrawal initiated a very efficient export of the chimeric protein (67). More recently, Liu and DeFranco (63) showed that a chimeric GR containing the IB␣ C-terminal NES displays a rapid and leptomycin B-sensitive nuclear export following hormone withdrawal.…”

The Nuclear Export Signal of Splicing Factor Uap56p Interacts with Nuclear Pore-associated Protein Rae1p for mRNA Export in Schizosaccharomyces pombe

“…HeLa Cell Nuclear Export Assay-Nuclear export assay in HeLa cells was performed according to the method described previously (25,26). DNA sequence coding for specific NES segments of interest was fused to glucocorticoid receptor (Gr) and GFP DNA sequences in Gr-GFP vector.…”

“…2D, panel b). However, in the presence of NES the fusion protein can exit the nucleus showing a diffused localization of the fusion protein throughout the cell, indicative of a functional NES (25). Fig.…”

The Nup153-Nup50 Protein Interface and Its Role in Nuclear Import

“…4C). Nuclear import was then assessed by tracking a cotransfected glucocorticoid-responsive import cargo, RGG (17). Using low levels of dexamethasone and tracking import in real time (Fig.…”