A combination of expansion microscopy and proximity labelling reveals conserved and unique asymmetric functional hubs at the trypanosome nuclear pore

Abstract

A nuclear pore consists of two outer rings flanking an inner ring connected to the nuclear envelope. Asymmetric structures at both nucleoplasmic and cytoplasmic faces moderate unidirectional transport of macromolecules. In the ancient trypanosomes, many of the pore (associated) proteins are not conserved. In particular, asymmetric components are mostly unknown, and the genome lacks orthologues to both, the nuclear mRNA-recruiting TREX-2 complex and the cytoplasmic mRNA remodelling complex Dbp5/Gle1. By combining expansion microscopy with proximity labelling, we mapped most of the 75 trypanosome nuclear pore localised proteins to specific subregions of the pore. At the nuclear site, we identified structural homologues to TREX-2 proteins, the Ran binding protein RanBPL and several trypanosome-unique proteins including NUP64 and NUP98. At the cytoplasmic site we found RanGAP, RanBP1 and the heterotrimeric NUP76 complex that we show to function in mRNA export, but no homologue to Dbp5/Gle1. Our novel, asymmetric and comprehensive map of the trypanosomes pore and its associated proteins supports a Ran-dependent mRNA export system with many trypanosome-unique features at both sides of the pore.