Abstract
BackgroundNuclear pore complexes (NPCs) are essential for facilitated, directional nuclear transport; however, the mechanism by which ~30 different nucleoporins (nups) are assembled into NPCs is unknown. We combined a genetic strategy in Saccharomyces cerevisiae with Green Fluorescence Protein (GFP) technology to identify mutants in NPC structure, assembly, and localization. To identify such mutants, a bank of temperature sensitive strains was generated and examined by fluorescence microscopy for mislocalization of GFP-tagged nups at the non-permissive temperature.ResultsA 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.ConclusionThe 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.
Highlights
Nuclear pore complexes (NPCs) are essential for facilitated, directional nuclear transport; the mechanism by which ~30 different nucleoporins are assembled into NPCs is unknown
Rationale for a genetic screen to identify temperature sensitive mutants that perturb NPC assembly and localization Work in our previous studies suggested that Green Fluorescence Protein (GFP)-based screening for NPC assembly mutants was a powerful approach
Mutants perturbing GFP-Nup localization could include cells with 1) NPC clusters in nuclear envelope (NE) subregions, 2) mislocalization of the GFPNup or aggregated NPC subcomplexes to the cytoplasm or nucleus, 3) mislocalization of the GFP-Nup due to indirect perturbation of the endoplasmic reticulum (ER) and NE membrane, or 4) decreased GFP-Nup incorporation followed by turnover resulting in cells with diminished total fluorescence
Summary
Nuclear pore complexes (NPCs) are essential for facilitated, directional nuclear transport; the mechanism by which ~30 different nucleoporins (nups) are assembled into NPCs is unknown. We combined a genetic strategy in Saccharomyces cerevisiae with Green Fluorescence Protein (GFP) technology to identify mutants in NPC structure, assembly, and localization. To identify such mutants, a bank of temperature sensitive strains was generated and examined by fluorescence microscopy for mislocalization of GFP-tagged nups at the non-permissive temperature. Eight spoke-like structures form the central NPC core and are flanked on the cytoplasmic and nuclear sides by ring structures. Yeast and vertebrate NPCs are responsible for identical transport reactions, and both structures are predicted to be functionally equivalent [9]
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