Importin 7 and importin alpha/importin beta are nuclear import receptors for the glucocorticoid receptor

Abstract

The vertebrate glucocorticoid receptor (GR) is cytoplasmic without hormone and localizes to the nucleus after hormone binding. GR has two nuclear localization signals (NLS): NL1 is similar in sequence to the SV40 NLS; NL2 is poorly defined, residing in the ligand-binding domain. We found that GR displayed similar hormone-regulated compartmentalization in Saccharomyces cerevisiae and required the Sxm1 nuclear import receptor for NL2-mediated import. Two metazoan homologues of Sxm1, importin 7 and importin 8, bound both NL1 and NL2, whereas importin alpha selectively bound NL1. In an in vitro nuclear import assay, both importin 7 and the importin alpha-importin beta heterodimer could import a GR NL1 fragment. Under these conditions, full-length GR localized to nuclei in the presence but not absence of an unidentified component in cell extracts. Interestingly, importin 7, importin 8, and importin alpha bound GR even in the absence of hormone; thus, hormonal control of localization is exerted at a step downstream of import receptor binding.

Figure 1.

Import of GR is dependent on hormone and two nuclear localization signals (NL1 and NL2) in yeast and mammalian cells. Bar, 10 μM for CV1 cell images, 1 μM for yeast images. (A) Diagram shows full-length GR with positions of the DNA-binding domain (DBD), ligand-binding domain (LBD), NL1, and NL2, other GR constructs used, and sequence similarities between GR and the SV40 large T-antigen. In K513-515A, lysines are mutated to alanines. (B) CV1 cells were transfected with pEGFP-N795. Twenty-four hours after transfection, cells were treated with EtOH (–H) or 1 μM Dex (+H) as indicated for 1 h. For hormone withdrawal, hormone-treated cells were incubated overnight in media lacking hormone. W303-1a yeast were transformed with pAdh-GR-GFP and grown overnight in selective media containing EtOH (–H) or 5 μM DAC (+H) as indicated; yeast were diluted to an OD595 of 0.1 and grown to OD595 of 0.4 before microscopy. For hormone withdrawal, treated cells were incubated in media lacking hormone for 1 h. The kinetics of GR nuclear transport are different in yeast than in mammalian cells; the cause of this kinetic difference has not been investigated. (C) CV1 cells were transfected and yeast cells transformed with noted plasmids and treated with EtOH (–H), 1 μM Dex (CV1 cells +H), or 5 μM DAC (yeast +H) as indicated.

Figure 2.

NL2-mediated import is altered in a Δsxm1 yeast strain. Bar, 1 μM. (A) Plasmids encoding FL GR K513-515A-GFP, N525-GFP, 540C-lacZ-GFP, and Lhp1 were transformed into PSY1200 (Δsxm1 strain) or PSY902 (parental control); as noted. Plasmid pPS1574 was cotransformed to ectopically express Sxm1p and yeast treated with either EtOH (–H) or 5 μM DAC (+H). (B) pAdh-N795 K513-515A-GFP and Pho4-GFP (EB0347) were transformed into pse1-1^ts or the control psy580 strain. (C) PSY1200 (red bars) or PSY902 (gray bars) were transformed with pAdh-N795-GFP or pAdh-N795-K513-515A-GFP and the pL2g3z GR reporter plasmid. For ectopic Sxm1p expression (blue bars), PSY1200 yeast were transformed with above plasmids and pPPS1117 expressing Sxm1p. Data represent the average values of six independent assays. Note: N795 K513-515A localization was unaffected in an Δsxm1 strain constructed in the Blobel lab (Rosenblum et al., 1997) in a different strain background than PSY1200 (Seedorf and Silver, 1997). These results suggest the presence of a genetic modifier, implying the existence of multiple import receptors for NL2 in some yeast strains.

Figure 3.

GR binds to importin 7 and importin 8. Input lane corresponds to 20% input. (A) GST, GST-importin 7 (7), GST-importin 8 (8), or GST-importin α (α) (4 μg) were immobilized on glutathione resin and incubated with 150 ng of purified rat GR for 2 h at 4°C. Bound proteins were resolved by SDS-PAGE and immunoblotted with rat GR-specific BuGR2 antibody. (B) Immobilized GST proteins in A were incubated with 12 ng in vitro transcribed and translated N795, N525, or 540C for 1 h at RT. Bound proteins were resolved by SDS-PAGE and detected by PhosphorImager. (C) Immobilized GST-BIB, GST NLS, GST-407-525, GST-407-525 K513-515A, or GST alone (4 μg) were incubated with 4 μg of purified importin 7 overnight at 4°C. Bound proteins were resolved by SDS-PAGE and immunoblotted with a polyclonal antibody to importin 7. (D) Substrates described in C were incubated with 72 μg of HeLa extract overnight at 4°C and immunoblotted as above. (E) Immobilized import receptors (4 μg) were preincubated with 40 μM RanQ69L for 1 h at 4°C and then incubated with 12 ng in vitro transcribed and translated full-length GR for 2 h at 4°C. (F) GST, GST-α, and GST-7 (4 μg) were immobilized on glutathione resin in the presence or absence of importin β overnight at 4°C. Bound import receptor complexes were incubated with 4 μg of a purified GR fragment (407–565) containing NL1 for 3 h at 4°C.

Figure 4.

Reconstitution of GR nuclear import in vitro. Bar, 10 μM. (A) GFP-GST-407-525 (0.5 μM) was incubated with permeabilized cells, 2 mg/ml HeLa extract, ATP mix, 6 μM RanQ69L, or apyrase for 30 min at 30°C or 4°C, as indicated. (B) Purified GR (30 nM) was incubated with digitonin-permeabilized cells, 2 mg/ml HeLa extract, ATP mix, 6 μM RanQ69L, or apyrase for 30 min at 30°Cor4°C, as indicated.

Figure 5.

Importin 7 and the importin α-importin β heterodimer import GR 407-525 in vitro. Bar, 10 μM. (A) BIB, SRP19, and SV40 import substrates (0.5–1 μM) and an ATP mix, 3 μM RanGDP, 6 μM RanQ69L, 2 μM importin 7, 2 μM importin 8, 4 μM importin α,or1 μM importin β, as indicated, were incubated with permeabilized cells as above. (B) Import assays were performed with 0.5 μM GFP-GST-407-525 and indicated import machinery. (C) Import assays were performed as above with 0.5 μM GFP-GST-407-525 K513-515A. (D) Import assays were performed with 1 μM SV40 NLS and importin 7.

Figure 6.

Importin 7 and the importin α-importin β heterodimer potentiate the import of full-length rat GR. Bar, 10 μM. (A) GR (30 nM), ATP mix, 3 μM RanGDP, and 2 μM importin 7, 2 μM importin 8, 4 μM importin α,or1 μM importin β, as indicated, were incubated with digitonin permeabilized cells for 30 min at 30°C. (B) GR (30 nM) was incubated with permeabilized cells, ATP mix, and the indicated concentration of HeLa cell extract. (C) GR (30 nM), ATP mix, 0.3 mg/ml HeLa extract and 2 μM importin 7 or 4 μM importin α were incubated with digitonin permeablized cells as above.

Figure 7.

Interaction of importin 7, importin 8, and importin α with GR is hormone independent. Input lane corresponds to 20% input. (A) GST, GST-importin 7 (7), GST-importin 8 (8), GST-importin α (α) were immobilized on glutathione-agarose and incubated with 12 ng of ³⁵S-labeled GR, which had been in vitro transcribed and translated in the presence or absence of 10 μM Dex. Bound proteins were resolved by SDS-PAGE and analyzed by PhosphorImager. (B) Immobilized import receptors were incubated with 240 μg of A549 cytosol in the presence or absence of 100 nM Dex for 30 min at room temperature. Bound proteins were resolved by SDS-PAGE and immunoblotted with the GR 283 polyclonal antibody.