Single-molecule measurements of importin α/cargo complex dissociation at the nuclear pore

Abstract

Macromolecules are transported between the cytoplasm and the nucleoplasm of eukaryotic cells through nuclear pore complexes (NPCs). Large (more than Ϸ40 kDa) transport cargoes imported into the nucleus typically form a complex with at least one soluble transport cofactor of the importin (Imp) ␤ superfamily. Many cargoes require an accessory cofactor, Imp ␣, which binds to Imp ␤ and to the nuclear localization sequence on the cargo. We previously reported the use of narrow-field epifluorescence microscopy to directly monitor cargoes in transit through NPCs in permeabilized cells. We now report an expanded approach in which singlemolecule fluorescence resonance energy transfer (FRET) is used to detect the disassembly of Imp ␣/cargo complexes as they transit through NPCs. We found that CAS, the recycling cofactor for Imp ␣, and RanGTP are essential for this dissociation process. After Imp ␣/cargo complex dissociation, most Imp ␣ and cargo molecules entered the nucleoplasm. In contrast, the majority of Imp ␣/cargo complexes that did not dissociate at the NPC in the presence of CAS and RanGTP returned to the cytoplasm. These data are consistent with a model in which Imp ␣/cargo complexes are dissociated on the nucleoplasmic side of the NPC, and this dissociation requires both CAS and RanGTP.FRET ͉ nuclear transport ͉ single-molecule fluorescence N uclear pore complexes (NPCs) mediate the bidirectional transport of proteins and RNAs across the doublemembrane nuclear envelope (NE) of eukaryotic cells. They are comprised of Ϸ30 different nuclear pore proteins (Nups), each present in an integer multiple of eight copies (1-3). The pore itself is Ϸ90 nm long and is Ϸ50 nm wide at its narrowest point. Flexible filaments extend out from the pore Ϸ50 nm into the cytoplasm, and a basket structure extends Ϸ75 nm into the nucleoplasm (1, 4). The pore is filled by natively unfolded (disordered) and highly flexible protein structures containing thousands of phenylalanine-glycine (FG) repeat motifs (3,(5)(6)(7)(8). This FG-network provides a selectivity filter, allowing small molecules (less than Ϸ20-40 kDa) to diffuse through and rejecting most larger molecules. Larger molecules (up to Ϸ25 MDa) penetrate the FG-network with the assistance of transport receptors of the importin (Imp) ␤ superfamily, which directly interact with the FG repeats (9-11). Import complexes consisting of Imp ␤, Imp ␣, and cargo are dissociated after transport by RanGTP, the GTP-bound form of the G protein Ran (12). Imp ␤ is recycled to the cytoplasm in a complex with RanGTP, and Imp ␣ is recycled in a complex with CAS and RanGTP. Activation of the Ran GTPase by the cytoplasmically located RanGAP frees Imp ␤ and Imp ␣ for another round of cargo transport (13-15; for recent reviews, see refs. 9-11).Numerous mechanisms of Imp ␣/cargo complex dissociation have been proposed. One possibility is that Imp ␣/cargo complexes dissociate spontaneously, and an autoinhibitory sequence in the N-terminal Imp ␤ binding domain of Imp ␣ inhibits cargo rebinding (16-18). Spo...

Nucleoporin Nup50 Stabilizes Closed Conformation of Armadillo repeat 10 in Importin α5

“…6, function a). In agreement with this idea, single molecule analysis of importin ␣/cargo complex dissociation at the NPC found that Nup50 alone is insufficient to promote importin ␣1/cargo complex dissociation on the time scale of transport (30). Instead, in vivo cargo displacement is mainly exerted by the combined action of CAS and RanGTP (30).…”

“…This idea is corroborated by the evidence that some of the sites bound by Nup50 in importin ␣1 are also critical for the assembly of CAS (27,29), the export receptor that, in concert with RanGTP, exports importin ␣ back to the cytoplasm. However, the putative role of Nup50 in dissociating the import complex was recently challenged by single-molecule observations in permeabilized cells that revealed Nup50 is insufficient to dissociate an importin ␣-NLS cargo complex on the time scale of nuclear transport (30). Finally, Nup50 was shown to be expressed in two splicing isoforms that play opposite roles in regulating the import reaction (31).…”

Self-regulated viscous channel in the nuclear pore complex

“…Fluorescent transiting substrates of different sizes (0.3-217 kDa in molecular weight, approximately 1.4-12.8 nm in diameter), including organic dyes, dextrans, proteins, transport receptor Imp β1, and the model import cargo complex Imp α/Imp β1/NLS-2xGFP (nuclear localization sequence of PPKKKRKV linked to a GFP dimer), were tracked through single native NPCs. Extensive control experiments have shown that labeled dyes do not alter functional nucleocytoplasmic transport of transport receptors and their cargo complexes (19,(20)(21)(22)(23). In 10 NPCs of 10 cells, 1,500 to 5,000 spatial locations for each substrate were collected from 110-750 singlemolecule nucleocytoplasmic transport events (Fig.…”

Choreography of importin-α/CAS complex assembly and disassembly at nuclear pores

“…We previously validated the smFRET approach for real-time measurements of nucleocytoplasmic transport (30). In short, positions of single molecules are detected by narrow-field epifluorescence microscopy and the oligomerization state of the donor-dye tagged protein vis-à-vis the acceptor-tagged protein is determined by FRET.…”

“…However, because transport occurs in the millisecond time regime, it is unclear to what extent these in vitro biochemical results relate to the rapid kinetics that occur during transport through intact NPCs. For example, although the nuclear basket nucleoporin Nup50 (Nup2 in yeast) catalyzes the dissociation of Imp-α/cargo complexes in vitro (25)(26)(27)(28)(29), CAS and RanGTP are also required for this reaction during real-time transport through intact NPCs (30).…”