The C-terminal nuclear localization signal of the sex-determining region Y (SRY) high mobility group domain mediates nuclear import through importin beta 1.
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.
Forwood
J K
JK
Nuclear Signaling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra City 2601, Australia.
Harley
V
V
Jans
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D005454:Fluorescence Polarization; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D021381:Protein Transport; D051381:Rats; D019849:Sex Determination Processes; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors; D028961:beta Karyopherins / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; C424166:KPNB1 protein, human; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D051876:Sex-Determining Region Y Protein; C497747:Sry protein, rat; D014157:Transcription Factors; D028961:beta Karyopherins; D004247:DNA