Cargo surface hydrophobicity is sufficient to overcome the nuclear pore complex selectivity barrier - PubMed (original) (raw)

Cargo surface hydrophobicity is sufficient to overcome the nuclear pore complex selectivity barrier

Bracha Naim et al. EMBO J. 2009.

Abstract

To fulfil their function, nuclear pore complexes (NPCs) must discriminate between inert proteins and nuclear transport receptors (NTRs), admitting only the latter. This specific permeation is thought to depend on interactions between hydrophobic patches on NTRs and phenylalanine-glycine (FG) or related repeats that line the NPC. Here, we tested this premise directly by conjugating different hydrophobic amino-acid analogues to the surface of an inert protein and examining its ability to cross NPCs unassisted by NTRs. Conjugation of as few as four hydrophobic moieties was sufficient to enable passage of the protein through NPCs. Transport of the modified protein proceeded with rates comparable to those measured for the innate protein when bound to an NTR and was relatively insensitive both to the nature and density of the amino acids used to confer hydrophobicity. The latter observation suggests a non-specific, small, and plant interaction network between cargo and FG repeats.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

Nuclear entry of BSA molecules modified by amino-acid side-chain analogues on their surface. The modifier used in each panel is denoted in superscript by the three-letter code of the corresponding amino-acid side chain it carries and the number indicates the degree of modification. (A–E) Confocal images of digitonin-permeabilized, nocodazole-treated HeLa cells 30 min after the introduction of a transport mixture containing innate or modified BSA (green) and a 66-kDa dextran (red) probe serving as an NE integrity marker. (F) Quantitative analysis of nuclear entry. Shown is the ratio between (background-corrected) nuclear and cytoplasmic mean fluorescence intensity (_F_nuc/_F_cyt) measured 30 min after introduction of innate BSA, BSA molecules modified by different amino-acid analogues, and BSA linked to SV40 large T-antigen NLS peptides (in the presence of cytosolic extract and an energy regenerating system). Data are presented as the mean±2 s.e.m. of determinations involving 20–30 cells for each column.

Figure 2

Figure 2

Inhibition of modified BSA transport by NPC blockers. (A–C) Confocal images of HeLa cells 30 min after the introduction of BSATrp4 either alone (A) or together with WGA (B) or αgp62 (C). The latter two are known blockers of NPC-mediated transport. Cells were permeabilized twice with the pore forming protein SLO, which is used for reversible permeabilization of the plasma membrane (Walev et al, 2001) (see Materials and methods) with the transport substrate introduced during the second permeabilization. (D) Quantitative analysis of nuclear entry. Shown is the ratio between (background-corrected) nuclear and cytoplasmic mean fluorescence intensity (_F_nuc/_F_cyt) measured 30 min after the second SLO permeabilization in which BSATrp4 was introduced to the cells. Data are presented as the mean±2 s.e.m. of determinations involving 6–12 cells for each column. Note that nuclear entry of the modified BSA in the absence of the blockers was as effective as that observed in digitonin-permeabilized cells.

Figure 3

Figure 3

Nuclear accumulation versus time traces of modified BSA. Shown are traces of BSA modified by (A) phenylalanine, (B) tryptophan, or (C) leucine analogues to varying degrees. Each data point represents the average obtained from multiple traces (see Figure 4C, for exact numbers) that were normalized before averaging. For convenience of presentation, error bars were not included (but see Figures 4A and 4C). The fits shown (solid lines) are provided only as guidance to the eyes.

Figure 4

Figure 4

Nuclear import kinetics of BSA modified by either hydrophobic amino acids or by NLSs and of an NTR. (A) A plot of _t_1/2 values measured for all BSA derivatives used in this study (blue circles) versus N—the estimated number of hydrophobic moieties attached to the protein. With our experimental setup and timeframe of the measurements, we could not accurately determine the nuclear entry rate of unmodified BSA because it was too low. It has been estimated that BSA traffics to the nucleus at least 600 times slower than transportin (Ribbeck and Gorlich, 2001). Combining this with our data, we estimate that native BSA enters the nucleus with a _t_1/2 of about 50 000 s, under the experimental conditions we used. This latter value is marked in the graph by a black square (note the break in the y axis). (B) Comparison between nuclear import kinetics of BSALeu66 and BSA derivatized by NLSs, which serve as substrates for the transport receptor complex importin α/β. In these experiments, the cells were supplemented by cytoplasmic extract and an energy regenerating system. (Inset) Nuclear import kinetics of BSALeu66 as compared with that of the (unloaded) nuclear import receptor transportin (fused to GST), both measured in unsupplemented cells. Data describing the nuclear import of the transport receptor were taken from Naim et al (2007). (C) _t_1/2 values derived for nuclear transport of BSA derivatives used in this study. The numbers in parenthesis denote the number of cells used in the analysis.

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