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

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 ␤ but not importin ␣. Using a range of quantitative binding assays including enzymelinked 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␤ independent of Imp␣. 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 ␤ with similar affinity to SRY. We also found that RanGTP, but not RanGDP, could dissociate the SRY-importin ␤ complex in solution using FP. We describe a novel double-fluorescent label DNA binding assay to demonstrate mutual exclusivity between importin ␤ 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␤ is of comparable affinity to that with which Imp␣/␤ recognizes conventional NLS-containing substrates.

Defective importin β recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations

“…Similar to the cNLS, the HMG domain was recognized poorly by IMP␣ (K d ϭ 112 nM), although IMP␤ recognition was slightly increased in the presence of IMP␣ (K d ϭ 2.4 nM). Thus, SRY joins a less common but growing list of arginine-rich NLScontaining proteins recognized by IMP␤ rather than IMP␣ (14,22,29,(31)(32)(33) that include the Rev (RqaRRnRRRRwR) from HIV type 1 (32, 34), the Rex protein of human T cell leukemia virus type 1 (mpKtRRRpRRsqRKRppt) (33), and the AP-1 family transcription factor CREB (26).…”

“…utable to residual IMP␤ bound to the nuclear envelope, which has been shown for a GFP-SRY HMG box (14) as well as the IMP␤-mediated nuclear import substrate IGFBP-3 (30).…”

“…In terms of nuclear architecture, the promoters of active genes are known to be in close proximity to the nuclear pores such that DNA in active chromatin may play an important role in effecting release of IMP␤ from SRY after transport through the nuclear pore. Analogous to GAL4 (37), DNA binding and IMP␤ binding by SRY seem to be mutually exclusive events (14), indicating that the NLSs of SRY may be regulated in part by DNA binding. Because the promoters of active genes are close to nuclear pore complexes in terms of nuclear architecture (11), it seems possible that after transport through the nuclear pore, DNA binding in conjunction with RanGTP may effect release of IMP␤ from SRY as has been hypothesized for GAL4 (37).…”

“…14). In terms of nuclear architecture, the promoters of active genes are known to be in close proximity to the nuclear pores such that DNA in active chromatin may play an important role in effecting release of IMP␤ from SRY after transport through the nuclear pore.…”

“…Either NLS is sufficient to target the large heterologous protein ␤-galactosidase (␤-gal) to the nucleus (13). Although the C-terminal NLS (cNLS) is recognized by the nuclear import receptor protein importin (IMP)␤1 (14), little is known regarding the nuclear import pathway mediated by the N-terminal NLS (nNLS); this NLS is not recognized by IMP␣ or -␤ (14). Both NLSs are highly conserved in SRY among mammals (Fig.…”