A genetic system to study the nuclear pore complex permeability barrier of the yeast Saccharomyces cerevisiae

Abstract

All nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs). To hinder uncontrolled mixing of cytoplasmic and nuclear content, NPCs maintain the permeability barrier that suppresses passage of inert macromolecules but allows facilitated translocation of even very large cargoes provided these are bound to nuclear transport receptors (NTRs). About a third of the proteins that built up the NPC contains so-called FG domains that are essential for establishing the NPC permeability barrier. NTRs bind to FG motifs and this interaction is essential for their privileged passage. How this binding accelerates NPC passage is one of the enigmas in the field. FG domains are quite diverse, di ffering in their cohesiveness, in their FG motifs, their length, their charge distribution, and in the sequence of the spacers between the individual FG motifs. The biophysical and biochemical properties of individual FG domains have been well studied in vitro. The signi cance of most of the observed features for the functionality of the permeability barrier in vivo, however, so far remained unclear. Previously, the in vivo signifi cance of individual FG domains was studied by generating S. cerevisiae deletion strains lacking multiple FG domains in various combinations. We show that previously reported lethal phenotypes of the combined deletion of FG domains were not exclusively caused by the deletions but were a result of the previously applied deletion strategy. Moreover, with an alternative deletion strategy, we show that S. cerevisiae tolerates more FG domain deletions than expected so far. The focus of this thesis has been to elucidate which FG domain features are essential for permeability barrier function in vivo. We established an in vivo system allowing to analyze the functional contribution of individual FG domains by testing the consequences of mutations, deletions, or exchange of these domains for cell viability. Employing this in vivo system, we find that the very cohesive FG domains of Nup100p and Nup116p play a prominent role in maintaining the permeability barrier. We show that not all FG domains can functionally replace these prominent FG domains, suggesting that NTR binding alone is not su cient to explain the functionality of the permeability barrier. Additionally, we show that the anchor points for FG domains within the NPC are not equivalent. Our findings support models of permeability barrier functionality that rely on the cohesiveness of FG domains. Based on our results, we, however, assume that both the excessive presence of cohesive FG mass as well as the absence of very cohesive FG domains is incompatible with viability.