Figure 6. Ubiquitination regulates NiV-M nuclear export.

(A) Ubiquitin depletion by MG132 treatment inhibits M nuclear export. HeLa cells were transfected with GFP-M alone (panels a and b), GFP-M plus HA-Ub (panel c) or GFP-M_bp1/2 (panel d). 24 hpt, cells were treated with 50 µM MG132 or DMSO as indicated, fixed 6 hrs later with 2% paraformaldehyde, stained with DAPI as well as a mouse anti-HA antibody followed by Alexa594-conjugated goat-anti-mouse secondary antibody to identify cells expressing HA-Ub, and imaged on a fluorescent microscope. Representative images are shown. (B) Ubiquitination patterns of wild-type M and the indicated mutants. HEK293T cells were co-transfected with HA-Ub (in which all the lysines were mutated to arginines to specifically look at monoubiquitination) and the indicated 3XFLAG-tagged M mutants or empty vector as control. M was immunoprecipitated as described in Materials and Methods and the ubiquitinated species were detected by immunoblotting using an anti-HA antibody. The banding patterns of K258A, K258R and M_bp2 were different from Mwt, whereas K263R was similar to Mwt. (C) Mimicking monoubiquitination restores nuclear export to K258R. One copy of ubiquitin was fused in frame to the C-terminus of 3XFLAG-K258R, and HeLa cells expressing K258R or K258R-Ub were stained with an anti-FLAG antibody. Quantification of the cytoplasmic/nuclear fluorescence intensity ratio for each mutant is shown in (D). There is significant difference between the localization patterns of K258R and K258R-Ub (p<0.0001, unpaired t test).