Kapinos LE et al. (2017), Karyopherins regulate nuclear pore complex barr...

XB-ART-54370

J Cell Biol 2017 Nov 06;21611:3609-3624. doi: 10.1083/jcb.201702092.

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Karyopherins regulate nuclear pore complex barrier and transport function.

Kapinos LE , Huang B , Rencurel C , Lim RYH .

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Nucleocytoplasmic transport is sustained by karyopherins (Kaps) and a Ran guanosine triphosphate (RanGTP) gradient that imports nuclear localization signal (NLS)-specific cargoes (NLS-cargoes) into the nucleus. However, how nuclear pore complex (NPC) barrier selectivity, Kap traffic, and NLS-cargo release are systematically linked and simultaneously regulated remains incoherent. In this study, we show that Kapα facilitates Kapβ1 turnover and occupancy at the NPC in a RanGTP-dependent manner that is directly coupled to NLS-cargo release and NPC barrier function. This is underpinned by the binding affinity of Kapβ1 to phenylalanine-glycine nucleoporins (FG Nups), which is comparable with RanGTP·Kapβ1, but stronger for Kapα·Kapβ1. On this basis, RanGTP is ineffective at releasing standalone Kapβ1 from NPCs. Depleting Kapα·Kapβ1 by RanGTP further abrogates NPC barrier function, whereas adding back Kapβ1 rescues it while Kapβ1 turnover softens it. Therefore, the FG Nups are necessary but insufficient for NPC barrier function. We conclude that Kaps constitute integral constituents of the NPC whose barrier, transport, and cargo release functionalities establish a continuum under a mechanism of Kap-centric control.

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Species referenced: Xenopus laevis
Genes referenced: napsa nup153 nup62 ran spr

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	Figure 1. RanGTP does not dissociate standalone Kapβ1 from the FG Nups. (A) Calculated solution binding isotherm for RanGTP/Kapβ1 at a ratio of 1.5:1 with Kd = 35 nM. (B) SPR response curves for RanGTP·Kapβ1 binding to different FG Nups followed by injections of Kapβ1 and RanGTP after a NaOH regeneration step. Vertical signals correspond to triple BSA injections that are used to measure FG Nup layer height. RU, resonance units. (C) Corresponding height changes to the FG Nup layer with respect to B. n = 10 per FG Nup. Error bars denote standard deviation.
	Figure 2. RanGTP dissociates Kapα from FG Nup–bound Kapα·Kapβ1. (A) Solution binding isotherm calculated for Kapα/Kapβ1 at a ratio of 1.5:1 with Kd = 210 nM. (B) SPR response curves for Kapα·Kapβ1 binding to different FG Nups show that Kapα is eluted by RanGTP. Vertical signals correspond to triple BSA injections that are used to measure FG Nup layer height. RU, resonance units. (C) Corresponding height changes to the FG Nup layer with respect to B. n = 10 per FG Nup. Error bars denote standard deviation.
	Figure 3. FG Nups bind Kapα·Kapβ1 more strongly than Kapβ1 and RanGTP·Kapβ1, which are similar. (A) FG Nup binding of Kapβ1 and related transport complexes are characterized by two distinct equilibrium dissociation constants at ∼0.1 µM (strong) and ∼10 µM (weak). In all cases, Kapα switches the Kapβ1 complex to a quantitatively higher binding affinity (lower Kd). RanGTP lowers the binding state to a value that is comparable with standalone Kapβ1. MG-NLS cargo does not significantly affect Kapα·Kapβ1 binding to Nup153 and Nup62. , P < 0.01; Student’s t test (see Table S3). (B) Kapα·Kapβ1 and MG-NLS·Kapα·Kapβ1 have a quantitatively higher binding affinity (lower Kd) than Kapβ1 when binding mixed FG Nups. To aid comparison, dashed lines at Kd values of 40 and 400 nM show that Kapα·Kapβ1 or MG-NLS·Kapα·Kapβ1 binds the FG Nups 10-fold stronger than standalone Kapβ1. **, P < 0.0001; Student’s t test. Box plots denote the median, first, and third quartiles. Error bars denote standard deviation, including outliers.
	Figure 4. FG Nup binding efficiency depends on MG-NLS/Kapα/Kapβ1 ratio, concentration, and RanGTP. Relative decrease of FG Nup–bound Kapβ1 due to RanGTP at different MG-NLS/Kapα/Kapβ1 ratios. A maximal reduction of Kapβ1 occurs when MG-NLS/Kapα/Kapβ1 = 10:10:1. Varying MG-NLS/Kapα/Kapβ1 ratios at constant CKapβ1 = 10, 100, and 1,000 nM enables comparisons between experiment (ΔRnorm; colored dots) and equilibrium calculations (ΔKapβ1; colored lines). For more information, see Fig. S5.
	Figure 5. Kapα facilitates the RanGTP-mediated release of Kapβ1. (A) Cartoon illustration of the experiment. (B) Immunofluorescence reveals that endoKapα and endoKapβ1 are retained and colocalize at the NE after permeabilization. Ran mix treatment effectively leads to a reduction in both endogenous pools. (C) ExoKapβ1 is not reduced by Ran mix after repopulating endoKap-reduced NPCs with 100 nM exoKapβ1. (D) Ran mix effectively reduces exoKapβ1 after repopulating endoKap-reduced NPCs with exoKapα·exoKapβ1 (Kapα/Kapβ1 = 10:1; CKapβ1 = 100 nM). (E) From D, exoKapβ1 retention at the NE after Ran mix (light red) shows qualitative agreement with equilibrium calculations (solid line) as a function of exoKapβ1 concentration. To aid comparison, these values were normalized by preRan mix values (dark red). (F) From D, exoKapα retention at NE before (dark green) and after Ran mix (light green). , P < 0.01; *, P < 0.001. Student’s t test (see Table S3). Box plots denote the median, first, and third quartiles. Error bars denote standard deviation, including outliers. Bars, 5 µm. In C and D, an endogenous protein-specific antibody that does not cross react with exoKapβ1 was used to immunostain for endoKapβ1. See Materials and methods for details.
	Figure 6. FG Nups are necessary but insufficient for NPC barrier function. (A) Cartoon illustration of all experiments. (B) Nuclear entry of MG and MG-NLS is prohibited in the presence of endoKapα and endoKapβ1 immediately after digitonin permeabilization. (C) Depleting endoKapα and endoKapβ1 by Ran mix abrogates NPC barrier function and facilitates the passive entry of MG and MG-NLS into the nucleus. (D) Adding back 100 nM exoKapβ1 sufficiently rescues NPC barrier function to prohibit MG and MG-NLS from entering the nucleus. (E) Fluorescence quantitation of MG in the nucleus at each of the above conditions. (F) Fluorescence quantitation of MG-NLS in the nucleus at each of the above conditions. n = 3 per experimental condition with a total of at least 26 cells each. ****, P < 0.0001; Student’s t test. Box plots denote the median, first, and third quartiles. Error bars denote standard deviation, including outliers. Bars, 5 µm.
	Figure 7. Kapβ1 turnover at NPCs is directly coupled to NLS-cargo release. (A) Cartoon illustration of all experiments. (B) Ran mix effectively reduces exoKapβ1 after repopulating endoKap-reduced NPCs with MG-NLS·exoKapα·exoKapβ1. (C) ExoKapβ1 retention at the NE after Ran mix (light red) shows qualitative agreement with equilibrium calculations (solid line) as a function of exoKapβ1 concentration. To aid comparison, these values were normalized by preRan mix values (dark red). (D) ExoKapα retention at NE before (dark green) and after Ran mix (light green). (E) Comparison of passive (dark purple) and Ran mix–activated (light purple) accumulation of MG-NLS in the nucleus. (F) Neither passive nor Ran mix–activated nuclear entry of MG-NLS was permitted in NPCs repopulated with exoKapβ1 (no exoKapα). ExoKapβ1 does not turn over under these conditions. (G) Fluorescence quantitation of nuclear MG-NLS before (dark purple) and after Ran mix (light purple) from F. n = 3 per experimental condition with a total of 10–12 cells each. , P < 0.01; **, P < 0.0001; Student’s t test. Box plots denote the median, first, and third quartiles. Error bars denote standard deviation, including outliers. Bars, 5 µm.
	Figure 8. Kapα-facilitated turnover of Kapβ1 softens the NPC transport barrier against nonspecific cargoes. (A) Cartoon illustration of the experiment. (B) Kapβ1 turnover is coupled to a softening of the NPC transport barrier with Ran mix–activated transport. (C) Fluorescence quantitation after Ran mix shows that MG entry into the nucleus is marginally increased. n = 3 per experimental condition with a total of at least 13 cells being analyzed each. ****, P < 0.0001; Student’s t test. Box plots denote the median, first, and third quartiles. Error bars denote standard deviation, including outliers. Bars, 5 µm.
	Figure 9. FG-centric versus Kap-centric NPC transport models. (Left) FG-centric models explain that an FG Nup barrier regulates selective transport through the NPC without invoking Kap occupancy. (Right) Kap-centric control argues that NPC barrier and transport function is regulated by Kaps in the pore. This is mediated by Kapα, which promotes NLS-cargo·Kapα·Kapβ1 import by switching the transport complex into a high-affinity FG Nup binding state. Upon reaching the nucleus, RanGTP switches Kapβ1 back to its lower affinity state while concomitantly releasing NLS-cargo and Kapα. At steady state, RanGTP·Kapβ1 export is sustained because it does not cross react with Kapα·Kapβ1 within the NPC. The model is constructed according to the equilibrium dissociation constants summarized in Table S4.

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