Karyopherin-independent spontaneous transport of amphiphilic proteins through the nuclear pore

Abstract

SummaryHighly selective nucleocytoplasmic molecular transport is critical to eukaryotic cells, which is illustrated by size-filtering diffusion and karyopherin-mediated passage mechanisms. However, a considerable number of large proteins without nuclear localization signals are localized to the nucleus. In this paper, we provide evidence for the spontaneous migration of large proteins in a karyopherin-independent manner. Time-lapse observation of a nuclear transport assay revealed that several large molecules spontaneously and independently pass through the nuclear pore complex (NPC). The amphiphilic motifs were sufficient to overcome the selectivity barrier of the NPC. Furthermore, the amphiphilic property of these proteins enables altered local conformation in hydrophobic solutions so that elevated surface hydrophobicity facilitates passage through the nuclear pore. The molecular dynamics simulation revealed the conformational change of the amphiphilic structure that exposes the hydrophobic amino acid residues to the outer surface in a hydrophobic solution. These results contribute to the understanding of nucleocytoplasmic molecular sorting and the nature of the permeability barrier.

Structural Mechanism of Nuclear Transport Mediated by Importin β and Flexible Amphiphilic Proteins

“…that demonstrated importin b-independent nuclear translocation of oversized proteins. These include albumin (66 kDa) with hydrophobic modifications on the surface (Naim et al, 2009;Ribbeck and Gö rlich, 2002), amphiphilic triple-helix of spectrin repeat (Kumeta et al, 2012), and importin a-and b-catenin, both of which contain amphiphilic helical repeats similar to HEAT repeats (ARM repeats; Fagotto et al, 1998;Koike et al, 2004;Wiechens and Fagotto, 2001). These lines of evidence strongly suggest that the hydrophobic/hydrophilic property of the protein is closely related to its passage through the NPC.…”

A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid

“…Numerous studies have provided evidence for deviations to this conventional nuclear import mechanism (reviewed by Wagstaff and Jans, 2009). For example, nuclear import of cyclin-B1-Cdc2 occurs independently of Ran but requires importin-β (Takizawa et al, 1999) and some nuclear import pathways are importin-β independent, for which the cargo acts as an NTR (Kumeta et al, 2012).…”

In vivo analysis of protein crowding within the nuclear pore complex in interphase and mitosis

“…To examine the effects of NTRs and their cargos on local protein crowding within the NPC, we performed in vitro transport assays as previously described 24, 25 . HeLa cells expressing GimRET-fused Nups were first treated with digitonin to permeabilize the cell membrane and remove cytoplasmic proteins.…”