Forwood Jade K, Kaur Gurpreet, Jans David A
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia.
Methods Mol Biol. 2007;390:83-97. doi: 10.1007/978-1-59745-466-7_6.
The sex-determining factor SRY plays an important role in male sexual development, diverting primordial gonads from the ovarian pathway toward male differentiation to form testes. SRY is a DNA-binding protein and gains access to the nucleus through two independently acting nuclear localization signals (NLSs) that flank the high mobility group (HMG) DNA-binding domain. We have reconstituted the nuclear import of SRY using an in vitro nuclear transport assay, showing that nuclear import of SRY can occur in the absence of additional exogenous cytosolic factors, with a significant reduction in nuclear transport in the presence of antibodies to the nuclear transport protein importin (Imp) beta1 but not Impalpha. We have also shown using in vitro binding assays that the C-terminal NLS of SRY binds directly to Impbeta1. Finally, we have shown that SRY can target green fluorescent protein to the nucleus in a mammalian transfected cell line; importantly, mutations known to result in sex reversal that map to either NLS impair nuclear accumulation implying that SRY nuclear import is critical to its function.
性别决定因子SRY在男性性发育中起着重要作用，它使原始性腺从卵巢发育途径转向男性分化，从而形成睾丸。SRY是一种DNA结合蛋白，通过位于高迁移率族（HMG）DNA结合结构域两侧的两个独立作用的核定位信号（NLSs）进入细胞核。我们利用体外核转运试验重建了SRY的核输入过程，结果表明，在没有额外外源性胞质因子的情况下，SRY也能发生核输入，当存在针对核转运蛋白输入蛋白（Imp）β1而非Impα的抗体时，核转运显著减少。我们还通过体外结合试验表明，SRY的C末端NLS直接与Impβ1结合。最后，我们证明SRY可以在哺乳动物转染细胞系中将绿色荧光蛋白靶向运输到细胞核；重要的是，已知导致性反转的突变定位于任一NLS，这会损害核积累，这意味着SRY的核输入对其功能至关重要。
