The main function of the nuclear pore complex (NPC) is to facilitate and regulate the transport between the cytosol and the nucleus but NPCs are also involved in various other cellular functions, including regulation of gene expression, translational control, signal transduction and cell differentiation. The nucleocytoplasmic transport system is composed of the NPC, that forms a large aqueous channel lined with FG-repeats containing nucleoporins (FG-Nups). FG-Nups constitute a permeability barrier, which prevents the passage of the majority of all proteins. Nuclear transport receptors (NTRs, also called importins, exportins or karyopherins) specifically recognize localization signals of cargo molecules and facilitate their passage through the central channel by transiently interacting with FG-Nups. Classical methods such as affinity purification or measurement of dissociation constants are not well-suited to globally identify NTR-cargo interactions because they are of a very transient nature, the spectrum of recognized cargos is huge and their dynamic concentration range comprises orders of magnitude. The exact cargo spectrum of the majority of NTRs, their specificity and even the extent to which active nucleocytoplasmic transport contributes to protein localization thus remains uncertain. To systematically map cargo-NTR relationships in an unbiased way in situ, I used proximity labeling mass spectrometry based on the so-called BioID system. I systematically fused BirA to various NTRs and other factors involved in nucleocytoplasmic transport. I found that at least one third of the human proteome is subject to active nuclear transport. I characterized the specific cargo spectrum of several NTRs and can thus estimate their specificity or overlap with other transport pathways. I identified the responsible transport pathways of various key protein complexes and demonstrated that those and components of pathways tend to be transported by related NTRs. The identification of the exact biotinylation sites provided evidence for the relevant interaction surfaces and sheds light in direct versus piggyback transport mechanisms.
To understand the compositional changes, potentially affecting the nucleocytoplasmic transport, within the NPC and NTR system better, I investigated them during development and within different cell types in Drosophila and Zebrafish. For the NPC only Nups, exposed to the environment, showed significant changes. The abundance changes of the NTRs were more dynamic and indicated a more flexible adaptation to changing circumstances
