Abstract
The sex-determining factor SRY is a DNA-binding protein that diverts primordial gonads from the ovarian pathway toward male differentiation to form testes. It gains access to the nucleus through two distinct nuclear localization signals (NLSs) that flank the high mobility group (HMG) DNA-binding domain, but the mechanisms through which these NLSs operate have not been studied. In this study, we reconstitute the nuclear import of SRY in vitro, demonstrating a lack of requirement for exogenous factors for nuclear accumulation and a significant reduction in nuclear transport in the presence of antibodies to importin beta but not importin alpha. Using a range of quantitative binding assays including enzyme-linked immunosorbent assay, fluorescence polarization, and native gel mobility electrophoresis, we assess the binding of importins to SRY, demonstrating a high affinity recognition (in the low nm range) by Imp beta independent of Imp alpha. In assessing the contribution of each NLS, we found that the N-terminal NLS was recognized poorly by importins, whereas the C-terminal NLS was bound by importin beta with similar affinity to SRY. We also found that RanGTP, but not RanGDP, could dissociate the SRY-importin beta complex in solution using FP. We describe a novel double-fluorescent label DNA binding assay to demonstrate mutual exclusivity between importin beta recognition and DNA binding on the part of SRY, which may represent an alternative release mechanism upon nuclear entry. This study represents the first characterization of the nuclear import pathway for a HMG domain-containing protein. Importantly, it demonstrates for the first time that recognition of SRY by Imp beta is of comparable affinity to that with which Imp alpha/beta recognizes conventional NLS-containing substrates.
Highlights
Sex determination and early gonadal differentiation of mammalian embryos take place around week 6 of development
To test the possibility that SRY(HMG)-GFP accumulated in the nucleus through its ability to diffuse through the nuclear pore complex (NPC) and bind to nuclear components, we performed the nuclear transport assay in the presence of the nuclear envelope-permeabilizing agent, CHAPS [43]
In this study we demonstrate that nuclear import of SRY is dependent on Imp, but not Imp␣, and that Imp is able to recognize SRY directly as demonstrated using a range of assays
Summary
Sex determination and early gonadal differentiation of mammalian embryos take place around week 6 of development. SRY1 (sex-determining region Y) is a DNA-binding protein encoded on the Y chromosome and acts as a genetic switch that diverts primordial gonads from the ovarian pathway toward male differentiation to form testes [1,2,3]. By binding the minor groove of DNA, SRY induces a large conformational change through helix unwinding, minor groove expansion, and DNA bending [12, 13] This is believed to be the molecular switch that allows distantly bound proteins of the transcription machinery to attain close proximity, thereby permitting interaction in a way that can influence transcription [12, 13]. In conventional NLS-dependent nuclear protein import, importin ␣ (Imp␣) recognizes the NLS and acts as an adapter to mediate binding of importin 1 (Imp) [17]. Direct evidence that transcription factors (TFs) may localize in the nucleus through this NLS-dependent importin ␣/-mediated nuclear transport pathway, is essentially restricted to inducible TFs such as those of the nuclear factor NF-B and STAT (signal transducer and activator of transcription) families [21,22,23]
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