PMC

Ca²⁺ chelation with EGTA inhibits CRT-dependent export without affecting nuclear import in permeabilized cells. (A) Diagram illustrating the assay for nuclear import (APC-NLS) and export (GR-GFP) in the same cells. (B) EGTA blocks CRT-dependent export of GR-GFP without affecting the nuclear import of APC-NLS in the presence of β-importin, Rch1, Ran, NTF2, NXT1, and RanBP1 (50μg/ml each). (C) Excess Ca²⁺ blocks CRT-dependent export of Rev-GFP without affecting the nuclear import of APC-NLS.

The low-affinity Ca²⁺ binding sites in the C-terminal domain of CRT are not essential for nuclear export activity. (A) Diagram of the CRT structure and sites of Ca²⁺ binding. (B to D) Export assays performed with CRT proteins containing deletions in the low-affinity Ca²⁺ binding C-terminal domain. CRT lacking the entire C-terminal domain retains its Ca²⁺-dependent inhibition of NES export, indicating that this is probably due to the high-affinity, low-capacity Ca²⁺ binding site. (E) EGTA-sensitive binding of CRT to the DBD is partially lost on removal of the C-terminal domain (residues 1 to 273). (F to H) Removal of the entire C-terminal domain (residues 1 to 273) from CRT abrogates the EGTA-sensitive export of GR. Although the C-terminal domain of CRT is not required for export, it is necessary for Ca²⁺ regulation of GR export.

Ran is a cofactor for NES-dependent export but not for DBD-dependent export. Nuclear export was assayed by supplementing digitonin-permeabilized cell assay mixtures with CRT (0.2 or 2.0 μM), Crm1 (0.2 μM), and different forms of Ran (1.9 μM each). NES-dependent export was assayed using a cell line that expresses a GFP fusion with Rev (RGG2.2) (), and DBD-dependent export was assayed using a cell line that expresses a GFP fusion with GR (3676) (). The Rev-GFP fusion also contains the ligand binding domain of GR, which imparts ligand-dependent nuclear import of the reporter protein. Prior to digitonin permeabilization, both cell lines were treated with Dex (1 μM) to induce nuclear import of the GFP reporters. (A and B) RanGTP stimulates Rev-GFP export in the presence of CRT or Crm1. (C and D) In contrast, neither WT nor MUT forms of Ran stimulate GR-GFP export in the presence of CRT or Crm1.

Ca²⁺ binding to CRT is necessary for nuclear export of GR. (A) Binding assay performed with GST-DBD or GST (2.5 μg each) immobilized on glutathione beads and CRT (500 ng). The amount of CRT bound in each reaction fraction was examined by immunoblotting for CRT. The CRT used in the binding assay was untreated, pretreated with 10 mM EGTA, or pretreated with 10 mM EGTA and 20 mM CaCl₂. Ca²⁺ removal from CRT inhibits binding to the DBD, and this can be reversed by addition of Ca²⁺. (B and C) Ca²⁺ is required for CRT-dependent GR export; however, Ca²⁺ inhibits CRT-dependent NES export. (D) The presence of excess EGTA and Ca²⁺ in the permeabilized-cell assay mixture does not affect the export mediated by Crm1. CRT and Crm1 were used at a final concentration of 1.1 μM each in the export assays. The pretreatment of CRT and Crm1 with EGTA is described in Materials and Methods.

Ca²⁺ depletion in vivo inhibits the nuclear export of GR. (A) Representative fields of cells expressing GR-GFP after induction of nuclear import with Dex (Import). Following Dex removal, the cells were treated for 5 h with vehicle (DMSO), Ion and BAPTA-AM, or TG and BAPTA-AM, and the GR-GFP distribution was recorded in living cells. (B) Measurements of the nuclear/cytoplasmic (Nuc/Cyto) ratios of GR-GFP fluorescence in cells incubated under conditions that deplete Ca²⁺. Depletion of luminal stores of Ca²⁺ with Ion andTG and chelation with BAPTA-AM results in significant reduction of GR export to the cytoplasm. (C) Depletion of luminal Ca²⁺ stores does not inhibit NES export. The RGG 2.2 cell line expressing Rev-GFP was treated with Dex to induce importing of the reporter, which concentrates in the nucleoli. The cells were then maintained for 6 h under conditions that deplete Ca²⁺. During the last 1 h, Dex was removed to allow Rev-GFP export to the cytoplasm. Rev-GFP export was observed whether or not Ca²⁺ was depleted.

Formation of export complexes involving CRT and Crm1. (A) The incorporation of RanGTP into complexes containing CRT and Crm1 was assayed using Ran preloaded with [γ-³²P] GTP. Target proteins were immobilized in microtiter wells (500 ng/well), and CRT or Crm1 (5 μg each) was added to each well together with 2 × 10⁴ cpm of radiolabeled RanGTP. Following incubation for 1 h at room temperature, the wells were washed four times and the bound fractions were released and assayed by scintillation counting. (B) RanGTP is not a cofactor for CRT binding to the DBD. Target proteins (1 μg each) were immobilized on glutathione beads, and CRT or Crm1 (500 ng each) was added to each sample in the absence or presence of Ran (1 μg) preloaded with cold GTP. The samples were mixed end over end for 2 h at room temperature, washed three times, eluted, and analyzed by immunoblotting with antibodies to CRT and Crm1. These data show that, like Crm1, NES recognition by CRT involves RanGTP. In contrast, DBD recognition by CRT does not involve RanGTP.

The DBD and hydrophobic NES use a common or overlapping binding site on CRT. (A) Binding assay with GST-DBD or GST (2 μg each) immobilized on glutathione beads and CRT (500 ng), RanGTP (2 μg), and WT or MUT PKI (2 μg) added in solution. The bound fractions were analyzed by immunoblotting for CRT. Including WT PKI in the reaction reduced the level of CRT bound to the DBD, indicating that these proteins bind to similar sites on CRT. This competition was not observed when RanGTP was omitted from the assay (data not shown), consistent with Ran acting as a cofactor for NES binding but not for DBD binding. (B) Nuclear export of GR-GFP was assayed in permeabilized cells using CRT (2.0 μM) in the presence of buffer, excess DBD (4.5 μM), or PKI (12 μM WT or MUT). (C) Competitive binding interactions between NES, DBD, and CRT measured in a biosensor assay. NES peptide was immobilized on the cuvette surface through a biotin-neutravidin linkage and was used to measure the Ran-dependent binding of CRT in the absence and presence of DBD in the solution. The proteins used in the assay were CRT (1.1 μM), Crm1 (1.1 μM), RanQ69L (1.9 μM), and WT and MUT DBD from GR (9.1μM each). CRT binds efficiently to NES peptide in the presence of Ran (green tracing), and this can be competed with the WT DBD (light blue tracing) but not with the transport-defective MUT DBD that contains the FF-to-AA mutations (dark blue tracing). Crm1 binding to the NES is unaffected by the presence of excess DBD (fuchsia tracing). (D) The DBD competes with NES in the CRT-dependent export pathway. The cell line expressing Rev-GFP was used to assay export in the presence of CRT (1.1 μM), WT PKI (12 μM), and WT DBD (9.1 μM) as indicated.