Figure 3. CRM1 cargo dissociation mechanism in fungi and animals.

(A) In fungi, cargoes are actively released from CRM1’s NES groove by formation of RanBP1-RanGTP-CRM1 trimeric complex. RanGTP-RanBP1 transiently released from CRM1 is catalyzed by RanGAP. In animals, RanBP1 strips RanGTP from CRM1, resulting in dissociation of NES cargo from CRM1. RanGAP catalyzes further dissociation of Ran-RanBP1. (B) GAP mediated RanGTP hydrolysis (arbitrary unit) at different time points. CRM1 and NES (PKI) inhibit RanGTP hydrolysis in both human (cyan) and yeast (orange). Addition of RanBP1 increases the rate of GAP hydrolysis. While addition of yCRM1 partially inhibit GAP mediated hydrolysis, addition of hCRM1 does not. Error bars represent standard deviation of quadruple repeats. (C) In vitro nuclear export of GST cargo in the presence of human or yeast CRM1/Ran/RanBP1 using semi-permeabilized HeLa cells. The reactions were stopped at different time points and level of nuclear cargo was stained with anti-GST antibody. (D) Quantification and statistical analysis of nuclear cargo intensity after normalization by DNA intensity. Error bars represent standard error of measurements for each set of data containing measurements from at least 30 cells. ** denotes p<0.01.

Figure 3—figure supplement 1. Low concentration of Triton-X prevents non-specific cytoplasmic binding and does not permeate the nuclear envelope.

(A) GST-NES-NLS was stained with anti-GST antibody. Control samples do not contain any reagents to prevent non-specific contamination. Images were collected at the same level of excitation and processed equally. 0.01% Triton-X works slightly better than 1% BSA in our hands. (B) PARP (poly [ADP-ribose] polymerase) could be stained when the cells were incubated in the presence of 0.25% Triton-X, but not 0.01% Triton-X.