The C-terminal Nuclear Localization Signal of the Sex-determining Region Y (SRY) High Mobility Group Domain Mediates Nuclear Import through Importin β1

Jade K. Forwood, Vincent Harley, David A. Jans

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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 β but not importin α. 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 Impβ independent of Impα. 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 β with similar affinity to SRY. We also found that RanGTP, but not RanGDP, could dissociate the SRY-importin β complex in solution using FP. We describe a novel double-fluorescent label DNA binding assay to demonstrate mutual exclusivity between importin β 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 Impβ is of comparable affinity to that with which Impα/β recognizes conventional NLS-containing substrates.

Original languageEnglish
Pages (from-to)46575-46582
Number of pages8
JournalJournal of Biological Chemistry
Issue number49
Publication statusPublished - 7 Dec 2001

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