A nuclear export signal is essential for the cytosolic localization of the Ran binding protein, RanBP1.

Abstract

Abstract. RanBP1 is a Ran/TC4 binding protein that can promote the interaction between Ran and 13-importin/13-karyopherin, a component of the docking complex for nuclear protein cargo. This interaction occurs through a Ran binding domain (RBD). Here we show that RanBP1 is primarily cytoplasmic, but the isolated RBD accumulates in the nucleus. A region COOH-terminal to the RBD is responsible for this cytoplasmic localization. This domain acts heterologously, localizing a nuclear cyclin B1 mutant to the cytoplasm. The domain contains a nuclear export signal that is necessary but not sufficient for the nuclear export of a functional RBD. In transiently transfected cells, epitope-tagged RanBP1 promotes dexamethasone-dependent nuclear accumulation of a glucocorticoid receptor-green fluorescent protein fusion, but the isolated RBD potently inhibits this accumulation. The cytosolic location of RanBP1 may therefore be important for nuclear protein import. RanBP1 may provide a key link between the nuclear import and export pathways.T RAFFIC between the nucleus and the cytoplasm occurs through nuclear pore complexes in the nuclear membrane. Proteins containing a nuclear localization signal (NLS) 1 are actively transported into the nucleus by the nuclear import machinery. Several components involved in this process have been identified, and nuclear protein transport can be reconstituted in permeabilized cells that have been depleted of cytoplasm (for review see . Nuclear protein export is not nearly as well understood, but the recent identification of nuclear export signals (NESs) may provide insight into the molecular basis for this process (for review see Gerace, 1995). NESs have been identified in three unrelated proteins thus far" protein kinase inhibitor (PKI) (Wen et al., 1995), HIV-1 Rev (Fischer et al., 1995), and heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) (Michael et al., 1995). The PKI and HIV-1 Rev NESs show limited similarity to one another, but the hnRNP A1 NES is distinctly different. All three proteins have been shown to shuttle between the nucleus and the cytoplasm and to mediate the export of other macromolecules.

Involvement of CRM1, a nuclear export receptor, in mRNA export in mammalian cells and fission yeast

“…5B). In agreement with a previous report (Richards et al 1996), we observed that RanBP1 is present exclusively in the cytoplasm (Fig. 5B, ¹LMB).…”

Isolated Mammalian andSchizosaccharomyces pombeRan-binding Domains RescueS. pombe sbp1(RanBP1) Genomic Mutants

“…Each of these proteins was subcloned with a T7 epitope tag in frame at its amino terminus (see MATERIALS AND METH-ODS). Consistent with previous studies (Richards et al, 1996;Zolotukhin and Felber, 1997), cells transfected with a construct expressing T7-tagged fulllength mouse RanBP1 showed largely cytoplasmic staining (Figure 2, left panels). In contrast, in cells expressing the T7-tagged mouse RBD, which lacks the NES motif (Figure 1), staining was largely nuclear (Figure 2, left panels).…”

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

“…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.…”