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. 2019 Apr 25;8:e41331. doi: 10.7554/eLife.41331

Figure 8. Mammalian nuclear transport system has higher efficiency.

(A) Nuclear export of GST-NES-NLS in the presence of yCRM1/yRan/yRanBP1 or their mutants (yCRM1T753Q, yRan, yRanBP1V150A and fused with mRanBP1’s NES). Protocol used in this experiment is similar as in Figure 3C. (B) Quantification of nuclear cargo intensity normalized by DNA in Figure 7E. Shown also includes unpaired student t-test between WT and mutant samples at each time point. Error bars represent standard error of measurements for each set of data containing measurements from at least 23 cells. Only 15 m and 30 m samples display statistical significances (** denotes p<0.01). (C) Representative images of GFP-NLS and mCherry-NES localization in human (HeLa) and yeast cells (W303.1a). pRS416 (for yeast transfection), pEGFP-C1 and pmCherry-C1 (for human transfection) plasmids were used to express fluorescent tagged NES or NLS proteins. mCherry-NES is constructed as mCherry-NESPKI-MBP-NLSSV40. GFP-NLS is constructed as GFP-NESPKI-MBP-NLSBPSV40. (D) Quantification and statistical analysis of nuclear cargo ratio in 8C.

Figure 8.

Figure 8—figure supplement 1. The NLS/NES used displayed similar affinities to yeast or human transport receptors.

Figure 8—figure supplement 1.

(A) Pull down shows that NES, NLS SV40 and NLSBPSV40 (that were used for Figure 8C) bind the yeast and human transport receptor with comparable efficiency. Kap60C and KPNA2C are the yeast and human importin α proteins with their N-terminal importin β binding (IBB) domains deleted. BPSV40 is same as reported in Hodel et al. (2001). (B) The different pull downs were performed at more concentrations to compare binding strength. More cytoplasmic localization of mCherry-NLS in human in theory could be due to tighter binding to CRM1 or weaker binding to human importin α (KPNA2). However, we showed that neither is true. In fact, SV40NLS binds to human importin α slightly stronger. Similarly, GFP-NLS localization difference was also not due to differences in binding to the transport receptors.
Figure 8—figure supplement 2. Yeast mCherry-NES-NLS were expressed at very low level compared to human cells.

Figure 8—figure supplement 2.

(A) Yeast sample was loaded 16x more than human sample but the yeast protein band intensity is still less than half of human protein band. Tot. proti. conc.: total protein concentration quantified by bicinchoninic acid assay (BCA). (B) antibodies against human Ran and CRM1 were not reactive against the yeast proteins. Proteins were purified from E. coli. (C) Estimated CRM1 and Ran concentrations in yeast and human cells.