FIG. 2.

Two nucleoporin-binding domains are required for TAP-mediated translocation of RNP cargoes across the NPC. (A) Schematic representation of the cat reporter gene encoded by plasmid pCMV128 (16). SD and SA indicate the splice donor and splice acceptor sites of the intron, respectively. (B) Human 293 cells were transfected with a mixture of plasmids encoding β-Gal (pCH110), CAT (pCMV128), and either GFP alone or GFP fused N-terminally to TAP or a TAP mutant, as indicated on the left. When indicated, a pGFP-N3 derivative encoding zzp15 was cotransfected (+p15, black bars). Cells were collected 48 h after transfection, and β-Gal and CAT activities were determined. CAT activities were normalized to the activities of the β-Gal internal control. The stimulation of cat gene expression by TAP mutants measured in three independent experiments is expressed as a percentage of the activity of wild-type TAP. The values are means ± standard deviations. (C) Protein expression levels were analyzed by Western blot with anti-GFP antibodies. The amount of cell extract loaded per lane was corrected for minor differences in transfection efficiencies, as revealed by the β-Gal internal control. The positions of TAP derivatives fused to GFP or of zzp15 are shown on the right. (D) Human 293 cells were transfected with the reporter plasmids pCMV128-RRE and pCH110 and plasmids encoding RevM10 fusions of TAP, TAP-1xUBA, or TAP-2xUBA. When indicated, a plasmid encoding zzp15 (+p15) was cotransfected (black bar). Thereporter plasmid pCMV128-RRE is identical to pCMV128 (panel A) but carries the HIV RRE inserted in the intron, downstream of the cat gene. The stimulation of cat gene expression by TAP mutants measured in three independent experiments is expressed as a percentage of the activity of RevM10-TAP coexpressed with p15. The values are means ± standard deviations. (E) Protein expression levels were analyzed by Western blot with anti-HA antibodies. The amount of cell extract loaded per lane was normalized to the activity of the β-Gal internal control. The positions of TAP derivatives fused to RevM10 are shown on the right. (F) RNase protection analysis was performed with cytoplasmic or total RNA fractions isolated from 293 cells cotransfected with pCMV128 and plasmid pCH110 encoding β-Gal. In lanes 3, 5, 8, and 10, plasmids encoding p15 and RevM10 or GFP fusions of TAP were cotransfected. As controls, the reporter plasmids were cotransfected with empty vectors expressing RevM10 or GFP alone together with the plasmid expressing p15 (lanes 2, 4, 7, and 9).