Dual nuclear import mechanisms of sex determining factor SRY: intracellular Ca2+ as a switch

The sex-determining region on the Y chromosome (SRY) has 2 nuclear localization signals (NLSs) that flank the DNA binding high mobility group (HMG) domain; the β-NLS and the CaM-NLS, which mediate nuclear transport through importin β1 (Impβ1) and the calcium-binding protein calmodulin (CaM), respectively. Here we reconstitute the nuclear import mediated by the 2 NLSs for the first time in vitro, establishing Ran independence of CaM-NLS-dependent transport. The β- and CaM-NLSs were found to be independently functional out of the context of the SRY HMG domain, dependent on Impβ1 and CaM binding, respectively. Intriguingly, direct protein binding assays also indicated competitive binding of Impβ1 and CaM to the SRY HMG domain. To assess the potential role of intracellular calcium in modulating SRY nuclear accumulation, Cos-7 cells expressing SRY and control constructs were treated with agents elevating or reducing intracellular Ca(2+) levels. The in vivo results, supported by experiments in vitro where transport was assessed with or without 2 μM Ca(2+), indicate a Ca(2+)-dependent mode of nuclear transport via the CaM-NLS/CaM, with inhibition of β-NLS/Impβ1-mediated nuclear import by intracellular Ca(2+). The results imply mutual exclusivity of nuclear transport via the 2 NLSs with intracellular Ca(2+) as the switch between the 2.