Molecular Determinants of Large Cargo Transport into the Nucleus

Abstract

Nucleocytoplasmic transport is tightly regulated by the nuclear pore complex (NPC), the “gatekeeper” of the nucleus. Even very large cargoes such as many pathogens, mRNAs and pre-ribosomal subunits can pass through the NPC intact. Such cargoes can typically only cross the permeability barrier if they are bound to nuclear transport receptors (NTRs), which recognize specific nuclear import and export signals (NLSs and NESs) on their surface. Compared to small import complexes, for large cargoes (>15 nm) there is very little quantitative understanding of the mechanism for efficient transport, the role of multivalent binding to NTRs via NLSs and effects of size differences. Here, we developed a set of four large cargo models suitable for nuclear transport studies, based on virus-like particles (capsids), in the size range of 17-36 nm and with tuneable numbers of up to 240 NLSs. Using permeabilized HeLa cells and a semi-automated imaging pipeline, we assayed nuclear import kinetics for a total of 30 large cargo models. We find that multivalent binding to NTRs can dramatically tune the import of the different capsids, with a minimum transport cutoff (of 10 NLSs in the lowest case) and a linear relationship between number of NLSs and import efficiency. In addition and taking advantage of the unprecedented cargo size range, we show that the requirements for transport scale non-linearly with size. A mathematical model based on fundamental energetics and kinetics considerations nicely recapitulates our experimental data, providing a physical framework to understand the limiting factors of nuclear transport. Together, our results reveal key molecular determinants on large cargo import kinetics in cells.