Isolation and characterization of new Saccharomyces cerevisiae mutants perturbed in nuclear pore complex assembly

A total of 121 mutant strains were isolated, with most showing GFP-Nic96 and Nup170-GFP mislocalized to discrete, cytoplasmic foci. By electron microscopy, several mutants also displayed an expansion of the endoplasmic reticulum (ER). Complementation analysis identified several mutant groups with defects in components required for ER/Golgi trafficking (sec13, sec23, sec27, and bet3). By directed testing, we found that mutant alleles of all COPII components resulted in altered GFP-Nup localization. Finally, at least nine unknown complementation groups were identified that lack secretion defects.The isolation of sec mutants in the screen could reflect a direct role for vesicle fusion or the COPII coat during NPC assembly; however, only those sec mutants that altered ER structure affected Nup localization. This suggests that the GFP-Nup mislocalization phenotypes observed in these mutants were the indirect result of overproliferation of the ER and connected outer nuclear envelope. The identification of potentially novel mutants with no secretory defects suggests the distinct GFP-Nup localization defects in other mutants in the collection will provide insights into NPC structure and assembly.Trafficking between the nuclear and cytoplasmic compartments is mediated by nuclear pore complexes (NPCs) [1,2]. These large, proteinaceous structures are embedded in a nuclear envelope (NE) pore formed by fusion of the inner and outer nuclear membranes. The three dimensional structure of NPCs from both Xenopus and budding yeast Saccharomyces cerevisiae is characterized by distinct substructures that are arranged with an 8-fold rotational symmetry [3]. Eight spoke-like structures form the central NPC core and are flanked on the cytoplasmic and nuclear sides by ring structures. Fibrils protrude from both the cytoplasmic and nuclear rings, with the nuclear fibrils gathered into a basket-like structure by a terminal ring. The global architectures of the yeast and vertebrate NPCs are hi