Figure 1 | Scientific Reports

Figure 1

From: Distinct mutations in importin-β family nucleocytoplasmic transport receptors transportin-SR and importin-13 affect specific cargo binding

Figure 1

Research workflow. (a) In addition to amino acid residues that contact cargoes in reported crystal structures, residues exposed on the concave surface facing the cavity of the TrnSR or Imp13 structure were considered candidate sites for mutational analysis. (b) Residues conserved across species within either TrnSR or Imp13 orthologs but not between them were identified via an ETA of 78 and 73 metazoan TrnSR and Imp13 sequences, respectively. The sequences were aligned, and the obtained amino acid frequencies of each column were used to estimate probabilities for use in computing the symmetric Kullback–Leibler divergence (KL value) for each aligned position (site n). (c) WT and mutagenized (mt) TrnSR and Imp13 (NTR) and their cargoes were prepared as bacterially expressed GST-mCherry-NTR fusion and GFP-cargo fusion proteins, respectively. Binding between the GST-mCherry-NTRs and the GFP-cargo was analyzed in all combinations by BHA. GST-mCherry-NTR was fixed on GSH-Sepharose beads and mixed with a bacterial extract containing the GFP-cargo protein. The beads were then observed with a confocal microscope, and the binding intensity was quantified from the fluorescence of mCherry and GFP in the images. (d) The cargoes were clustered based on their NTR mutant-binding specificity.

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