Yeast nuclear pore complex assembly defects determined by nuclear envelopereconstruction

Assembly of nuclear pore complexes (NPCs) is a critical yet poorly understo od cellular function. One approach to studying NPC assembly is to identify yeast mutants defective in this process. This requires robust assays for NP C assembly that can be used for phenotypic analysis. We have previously rec onstructed yeast nuclei from electron micrographs of serially sectioned cel ls to precisely determine the number of NPCs (Winey ct al, 1997). Here we r eport the analysis of strains mutant in either of two nucleoporin-encoding genes, NIC96 (Zabel ct al, 1996) and NUP192 (Kosova ct al, 1999), Using con ditional alleles of either gene, we have found that the NPC number falls si gnificantly following shift to the restrictive temperature. We conclude tha t the drop in NPC number results from the failure to assemble new NPCs duri ng cell divisions, leading to the dilution of NPCs that existed when the ce lls were shifted to the restrictive temperature. We are also able to docume nt a subtle defect in NPC numbers in nup192-15 cells at their permissive te mperature. The data presented here quantitatively demonstrate that NPC numb ers fall in nic96-1 and nup192-15 strains upon shifting to the restrictive temperature, indicating that these gene products are required for NPC assem bly, (C) 2000 Academic Press.