AbstractThe nuclear pore complex (NPC) is an 8-fold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ~30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure, however, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed Nuclear Rim Intensity Measurement or NuRIM, to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188 that their altered expression leads to significant changes in Nup stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.SignificanceThe nuclear pore complex (NPC) is one of the largest protein complexes in eukaryotes comprising over 500 nucleoporin subunits. The NPC is essential for transport of biomolecules across the nuclear envelope, however, due to its enormous size, it has been a challenge to characterize its molecular architecture. Herein, we have developed a novel, widely applicable imaging pipeline to determine the absolute nucleoporin abundances in native yeast NPCs. This reveals that the NPC composition dramatically differs between yeast and human despite an overall conservation of individual subunits. We also applied our imaging pipeline to examine yeast mutants revealing a remarkable compositional plasticity of NPCs. Our stoichiometry analyses provide an important resource for the generation of high-resolution structure models of the NPC.
Identifying Protein Complexes in the Nuclear Envelope by Fluorescence Fluctuation Microscopy
