Yeast Nuclear Pore Complex Assembly Defects Determined by Nuclear Envelope Reconstruction

Assembly of nuclear pore complexes (NPCs) is a critical yet poorly understood cellular function. One approach to studying NPC assembly is to identify yeast mutants defective in this process. This requires robust assays for NPC assembly that can be used for phenotypic analysis. We have previously reconstructed yeast nuclei from electron micrographs of serially sectioned cells to precisely determine the number of NPCs (Winey et al.,1997). Here we report the analysis of strains mutant in either of two nucleoporin-encoding genes, NIC96(Zabel et al.,1996) and NUP192(Kosova et al.,1999). Using conditional alleles of either gene, we have found that the NPC number falls significantly following shift to the restrictive temperature. We conclude that the drop in NPC number results from the failure to assemble new NPCs during cell divisions, leading to the dilution of NPCs that existed when the cells were shifted to the restrictive temperature. We are also able to document a subtle defect in NPC numbers in nup192-15cells at their permissive temperature. The data presented here quantitatively demonstrate that NPC numbers fall in nic96-1and nup192-15strains upon shifting to the restrictive temperature, indicating that these gene products are required for NPC assembly.