FIGURE 5.

The N-terminal fragment of importin β containing 297 amino acids (N297) inhibits ERAD of NHK. A, effects of C-terminal truncation mutants of importin β on NHK ubiquitination in vitro. The diagram represents recombinant proteins used in this experiment. WT importin β (1 μg) or its truncation mutants N603 (0.7 μg) or N297 (0.35 μg) was used as indicated. Apyrase was added to deplete ATP. B, quantification of NHK ubiquitination in A. The data represent the means ± S.E. of three independent experiments. The relative intensity of polyubiquitin smear (Ubn-NHK) in reaction with control cytosol for 1 h (lane 8) was set to 1. Ran binding was quantified from supplemental Fig. S6B. C, N297 overexpression causes accumulation of NHK in cells. HEK293 cells steadily expressing NHK-HA were transfected with plasmids encoding WT importin β or its mutant N297 as indicated. 24 h later, the cells were treated with MG132 as indicated for 6 h and then analyzed by IB. D, effect of N297 overexpression on association of importin β with ER. Microsomes were prepared from HEK293 cells transfected with plasmids encoding WT importin β or its mutant N297 as indicated. A shorter exposure (shorter) and a longer exposure (longer) images were shown for importin β blotting. Imp, importin.