Specificity and mechanism of 1,6 hexanediol-induced disruption of nuclear transport

Abstract

Selective transport through the nuclear pore complex (NPC) depends on the dynamic binding of the intrinsically disordered components of the NPC, the FG-nups, with each other and with nuclear transport receptors (NTRs). Hydrophobic interactions with the phenylalanines of FG-nups are critical for this dynamic binding. 1,6-hexanediol (1,6HD), is an aliphatic alcohol that interferes with hydrophobic interactions. Here we assessed the specificity and mechanism by which 1,6HD disrupts the permeability barrier of NPCs in live bakers yeast cells. Exposure to 1,6HD (10 min, 0-5%) leads to gradual loss of the NPC permeability. This is likely a direct effect on the nuclear transport machinery as cell viability, the pH and ATP levels in the cytosol, as well as the appearance of mitochondria, Golgi, peroxisomes, ER, vacuoles, plasma membrane, nucleolus, secretory pathway and stress granules are not notably changed. There are however effects on the cytoskeleton and Hsp104 to be noted. While 1,6HD treatment does not lead to dissociation or degradation of NPC subunits, a massive relocation of multiple NTRs from NPCs does occur. This displacement quantitatively correlates with the increased passive permeability of NPCs. The loss of NTRs and associated cargo will present a major change in the macromolecular crowding and composition and hence the physicochemical properties of the central channel. We conclude that 1,6HD provides a surprisingly specific intervention to temporarily permeate NPCs and we present evidence that the mechanism includes release of NTRs from the NPCs.

Nucleoporin Nsp1 surveils the phase state of FG-Nups

“…The focus is too small to assess using FRAP, and hence we studied if exposure of cells to 1,6-hexanediol would dissolve it. 1,6-Hexanediol is an aliphatic alcohol and 10 min exposure to 5% 1,6-hexanediol provides a surprisingly specific disruption of the interactions between FG-Nups and NTRs 69 and between FG-Nups 41,[43][44][45][46] , and generally dissolves liquid-like compartments 70 . Indeed, the exposure to 1,6hexanediol reduced the frequency of observing the focus in the emerging daughter from 65% to less than 3%, strongly indicating that the Nsp1 focus is a phase-separated, liquidlike condensate (Figure 2EF).…”