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. 2014 May 15;7(6):2768-79.
eCollection 2014.

Characterization of karyopherins in androgen receptor intracellular trafficking in the yeast model

Minh M Nguyen  1 , Robert M Harmon  2 , Zhou Wang  1
Affiliations

Characterization of karyopherins in androgen receptor intracellular trafficking in the yeast model

Minh M Nguyen et al. Int J Clin Exp Pathol. .

Abstract

Background: Mechanisms regulating androgen receptor (AR) subcellular localization represent an essential component of AR signaling. Karyopherins are a family of nucleocytoplasmic trafficking factors. In this paper, we used the yeast model to study the effects of karyopherins on the subcellular localization of the AR.

Methods: Yeast mutants deficient in different nuclear transport factors were transformed with various AR based, GFP tagged constructs and their localization was monitored using microscopy.

Results: We showed that yeast can mediate androgen-induced AR nuclear localization and that in addition to the import factor, Importinα/β, this process required the import karyopherin Sxm1. We also showed that a previously identified nuclear export sequence (NES(AR)) in the ligand binding domain of AR does not appear to rely on karyopherins for cytoplasmic localization.

Conclusions: These results suggest that while AR nuclear import relies on karyopherin activity, AR nuclear export and/or cytoplasmic localization may require other undefined mechanisms.

Keywords: Androgen receptor; karyopherin; nucleocytoplasmic trafficking.

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Figures

Figure 1
Figure 1
Constructs used to test the ability of NESAR and AR localization in yeast. All sequences were cloned into yeast p416 GPD expression vector containing a URA3 marker and GPD promoter for constitutive expression. A. Construct of GFP-tagged full length human androgen receptor and will be referred to as GFP-AR. B. Construct containing two GFPs and NLS from SV40 used as a nuclear localization control and will referred to as 2GFP-NLSSV40. C. Construct with two GFPs, NLS from SV40 and nuclear export signal from protein kinase inhibitor (NESPKI) used for cytoplasmic localization control and will be referred to as 2GFP-NLSSV40-NESPKI. D. Construct with two GFPs, NLS from SV40 and nuclear export sequence from AR and will be referred to as 2GFP-NLSSV40-NESAR. Constructs are not drawn to scale. GFP, green fluorescent protein; NLS, nuclear localization signal; NES, nuclear export signal. SV40, Simian vacuolating virus 40.
Figure 2
Figure 2
Yeast can mediate ligand dependent AR nuclear localization. Wild-type yeast strain (BY4741) was transformed with GFP-AR and grown in the absence (-DHT) or the presence (+DHT) of ligand. Nuclei were stained with Hoechst. The localization of GFP-AR and Hoechst staining was determined by fluorescence microscopy. In the absence of ligand, GFP-AR is evenly distributed in wild-type yeast. In the presence of ligand, GFP-AR is localized to the nucleus. Red arrows indicate yeast nuclei. DHT, 5α-dihydrotestosterone.
Figure 3
Figure 3
Sxm1 is required for ligand dependent nuclear localization of AR. sxm1Δ yeast strain was transformed with GFP-AR alone or GFP-AR and a WT copy of Sxm1 (+Sxm1) and grown in the presence ligand (+DHT). Nuclei were stained with Hoechst. The localization of GFP-AR and Hoechst staining was determined by fluorescence microscopy. In the sxm1Δ mutant, the ligand induced nuclear localization that was observed in wild-type yeast was abrogated. When a wild-type copy was reintroduced into the sxm1Δ mutant, ligand dependent nuclear localization of GFP-AR was restored. Red arrows indicate yeast nuclei. Sxm1, Suppressor of mRNA export mutant.
Figure 4
Figure 4
NESAR abrogates NLSSV40 nuclear localization: Wild-type yeast strain BY4741 was transformed with the indicated constructs and nuclei were stained with Hoechst. GFP localization and Hoechst staining were viewed using fluorescence microscopy. Yeast transformed with 2GFP-NLSSV40 displayed nuclear localization. Yeast transformed with 2GFP-NLSSV40-NESPKI displayed cytoplasmic localization. Yeast transformed with 2GFP-NLSSV40-NESAR abrogated NLSSV40 nuclear localization but did not display strong cytoplasmic GFP localization as that seen in 2GFP-NLSSV40-NESPKI. Red arrows indicate yeast nuclei. PKI, protein kinase inhibitor.
Figure 5
Figure 5
Crm-1 mutant does not affect cytoplasmic localization of 2GFP-NLSSV40-NESAR. Yeast crm1-1 heat sensitive mutant strain was transformed with either 2GFP-NLSSV40-NESPKI or 2GFP-NLSSV40-NESAR. Nuclei were stained with Hoechst. GFP localization and Hoechst staining was viewed using fluorescence microscopy. At permissive temperature, (25°C) 2GFP-NLSSV40-NESPKI and 2GFP-NLSSV40-NESAR are both localized in the cytoplasm. However, at non-permissive temperature (37°C), 2GFP-NLSSV40-NESPKI was found in the nucleus, while 2GFP-NLSSV40-NESAR localization remained unchanged. Red arrows indicate yeast nuclei.
Figure 6
Figure 6
Other export karyopherins do not affect NESAR localization. Yeast msn5Δ and los1Δ deletion mutants were transformed with the indicated constructs. Nuclei were stained with Hoechst. GFP localization and Hoechst staining was viewed using fluorescence microscopy. The localization of the GFP fusion proteins in (A) msn5Δ or (B) los1Δ was indistinguishable from that found in wild-type yeast. Red arrows indicate location of nuclei.
Figure 7
Figure 7
Import karyopherins do not affect NESAR localization. Yeast import karyopherin mutants were transformed with the indicated constructs. Nuclei were stained with Hoechst. GFP localization and Hoechst staining was viewed using fluorescence microscopy. Shown here is yeast kap123Δ deletion mutant strain as representative of other import mutants. The localization of the GFP fusion proteins in these import mutants was indistinguishable from that found in wild-type yeast. Red arrows indicate location of nuclei.
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