Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking

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Abstract
Nuclear pore complexes selectively transport cargoes across the nuclear envelope. Now, two studies published in this issue follow the movement of single cargo proteins (Lowe et al.) and mRNA transcripts (David Grünwald and Robert Singer) as they translocate across the nuclear envelope, with nanometre precision and high temporal resolution. They discover previously unidentified transport sub-steps and provide fundamental insight into the mechanism of selective transport through the nuclear pore complexes. Nuclear pore complexes selectively transport cargos across the nuclear envelope. Here, a nuclear transport assay has been developed that allows the movement of single cargo proteins to be followed in real time. A succession of transport substeps is observed, and the NPC is found to be functionally asymmetric to importing cargos. The study provides insight into the mechanism of selective transport through the NPC. The nuclear pore complex (NPC) mediates all exchange between the cytoplasm and the nucleus. Small molecules can passively diffuse through the NPC, whereas larger cargos require transport receptors to translocate1. How the NPC facilitates the translocation of transport receptor/cargo complexes remains unclear. To investigate this process, we tracked single protein-functionalized quantum dot cargos as they moved through human NPCs. Here we show that import proceeds by successive substeps comprising cargo capture, filtering and translocation, and release into the nucleus. Most quantum dots are rejected at one of these steps and return to the cytoplasm, including very large cargos that abort at a size-selective barrier. Cargo movement in the central channel is subdiffusive and cargos that can bind more transport receptors diffuse more freely. Without Ran GTPase, a critical regulator of transport directionality1, cargos still explore the entire NPC, but have a markedly reduced probability of exit into the nucleus, suggesting that NPC entry and exit steps are not equivalent and that the pore is functionally asymmetric to importing cargos. The overall selectivity of the NPC seems to arise from the cumulative action of multiple reversible substeps and a final irreversible exit step.

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