Figure 7.

The importin β binding domain acts as an NLS. (A) SREBP-2 (343–460) is sufficient for nuclear import in living cells. Purified recombinant His-SREBP-2(1–370) (a) or GFP-SREBP-2(343–460) (b) was injected into the cytoplasm of HeLa cells. After incubating for 30 min at 37°C, the cells were fixed, and the localization of each protein was examined by indirect immunofluorescence using anti-human SREBP-2 antibodies (a) or directly by fluorescent microscopy (b). (B) SREBP-2 (343–460) exactly reproduces the nuclear import of full-length SREBP-2 in vitro. Permeabilized HeLa cells were incubated with 10 μl of reaction mixture containing 6.5 pmol of GFP-SREBP-2(343–460) in the absence (a) or the presence (b and c) of recombinant transport factors: (b) 4 pmol of importin β; (c) 4 pmol of importin β, 4 pmol of Ran, and 3 pmol of p10/NTF2. All reactions contained an ATP regeneration system and 0.5 mM GTP. Import reactions were performed as in Figure 1B and examined by fluorescent microscopy.