Kinetic parameters for nuclear uptake of β-gal fusion proteins in mechanically perforated (a and b) and microinjected (c) HTC cells. (a) Results shown are from a single typical experiment representative of a series of similar experiments where each data point represents six separate measurements of each Fn and Fc with background subtracted, where the SEM for the individual measurements was not more than 12% of the value of the mean. Data were fitted for the function Fn/c = Fn/cmax (1 - e - kt), where Fn/cmax is the maximal level of nuclear accumulation, k is the rate constant, and t is the time in min. The time at which nuclear accumulation is half-maximal (t_1/2) is 12 ± 3.7 min for wild-type (wt) SRY nNLS-β-gal in the presence of cytosol. Nuclear accumulation of a conventional IMPα/β-recognized large-tumor antigen-NLS-β-gal fusion protein in the same system (not shown) gave an Fn/cmax of >4 on the same day, with accumulation half-maximal at 6 min. In contrast, β-gal alone was excluded from the nucleus (Fn/cmax of ≈0.4, not shown). (b) The t_1/2 for wild-type SRY cNLS-β-gal is 10.5 ± 3.1 and 8.3 ± 1.7 min in the presence or absence of cytosol, respectively. CLSM images are shown for cells after 35 min of incubation. (c) In vivo studies of nuclear uptake of wild-type SRY nNLS-β-gal and cNLS-β-gal, compared with that of β-gal alone, in microinjected HTC cells. The t_1/2 values for wild-type SRY nNLS-β-gal and cNLS-β-gal nuclear accumulation were 1.2 and 1.7 min, respectively.

A model for nuclear import of SRY from normal males and XY females. The distinct NLSs of SRY use different import pathways. The cNLS of SRY is recognized by IMPβ, which docks the transport complex at the nuclear pore complex (NPC) followed by translocation through it. After nuclear entry of the complex, RanGTP binds to IMPβ to trigger release of SRY; DNA binding by SRY may also facilitate the release process. The SRY nNLS can mediate nuclear import through a novel pathway not utilizing conventional nuclear import factors such as IMPs but an unidentified transport factor, TF, possibly calcium-calmodulin. In the XY females carrying the R75N and R76P mutations, the nNLS-dependent nuclear import pathway is probably nonfunctional, but the cNLS pathway is most likely active because both mutants bind IMPβ normally. In the XY female carrying the cNLS mutation, R133W, SRY binds IMPβ with reduced affinity due to the direct effect of the mutation; the nNLS pathway is probably functional. In the XY female carrying the R62G nNLS mutation, reduced IMPβ binding may imply that both pathways are impaired, with the residual R62G protein that is translocated to the nucleus unable to bind DNA efficiently (similar to the R75N mutation), or bend DNA to the correct angle to establish the correct architecture in the chromatin.

(a) Sequence conservation of the NLSs of SRY HMG domains. The nNLSs and cNLSs are highly conserved with basic residues (in bold) almost invariant between species. Arrows indicate the position of SRY mutations in four patients with XY gonadal dysgenesis; each substitution was of conserved arginines (R62G, R75N, R76P, and R133W). (b) Immunostaining of wild-type and mutant SRY proteins. COS-7 cells were transiently transfected with plasmids encoding wild-type or mutant FLAG epitope-tagged SRY. Mutant proteins identified in XY gonadal dysgenesis patients demonstrated partly cytoplasmic staining, whereas wild-type SRY showed strictly nuclear staining. Images were derived by using CLSM at ×60 magnification 2 days posttransfection. (c) Extent of nuclear accumulation of SRY mutants over a 3-day period. CLSM images were analyzed to determine the Fn/c. Results (mean ± SEM, for n > 22) relative to SRYwt (Fn/c of 15) are shown for SRYR62G, SRYR133W, and SRYR62G/R133W compared with SRYwt in one series (Left) and R75N and R76P to SRYwt in another (Right). Significant differences between wild type and mutants are indicated by ^* (P < 0.05), ^** (P < 0.01), and ^*** (P < 0.001).

(a) DNA binding of SRYwt, SRYR62G SRYR133W, and SRYR62G/ R133W. (Upper) An autoradiograph of in vitro-translated ³⁵S-labeled SRYwt, SRYR62G and SRYR133W proteins analyzed by 12% SDS/PAGE, showing a single 30-kDa band. Luc, luciferase (negative control). (Lower) A typical single EMSA experiment demonstrating SRYwt, SRYR62G, and SRYR133W (860 pg) binding to the SRY DNA-binding consensus site AACAAT. Luciferase (lane 1) is included as a negative control. Products of the binding assay were analyzed on a 6% native polyacrylamide gel in 0.5× TBE buffer (90 mM Tris/64.6 mM boric acid/2.5 mM EDTA, pH 8.3), dried, and autoradiographed for 5 h at -70°C. SRYwt and SRY-R133W proteins form DNA complexes (arrow), but SRY-R62G does not. (b) DNA bending by wild-type and R62G and R133W mutant SRY HMG domains. Circularly permuted probes (a to g) were labeled with ³²P and incubated with ≈100 ng of R62G and 4 ng of R133W and wild-type HMG domains. Bend angles were estimated as described (27). (c Top). The HMG box of SRY is an 80-aa DNA-binding domain, comprising three α-helices (numbered in red) that form an L conformation. Note that the DNA (green) is severely bent and unwound after binding (35). The nNLS is formed by the extended chain at the N-terminal end of the HMG box and part of helix 1 and is predicted to be in close proximity to the cNLS. The lower three panels show close-up views of arginine side chains R62, R75, and R76 (in light blue) substituted in cases of XY sex reversal. Although R62 and R75 contact DNA, R76 does not. (d) EMSA analysis of SRY binding to DNA in competition with IMPα/β. SRY HMG box (40 ng) was incubated with 0, 20, 40, 60, 80, or 100 ng of IMPα/β (lanes 2–7). Protein–DNA complexes (arrow) were resolved from free DNA on nondenaturing 6% polyacrylamide Tris/borate/EDTA gels.