A ROLE FOR THE DIVERGENT ACTIN GENE, ACT2, IN NUCLEAR-PORE STRUCTURE AND FUNCTION

We have identified a temperature-sensitive allele of the yeast: diverg ent actin gene ACT2, act2-1, which displays defects in nuclear pore co mplex (NPC) structure and nuclear import at the restrictive temperatur e. Although defective in nuclear import, act2-1 cells still selectivel y retain reporter proteins in the nucleus, and by indirect immunofluor escence the actin cytoskeleton appears normal. Previous studies in Aca nthamoeba and Saccharomyces cerevisiae reported that the cellular loca tion of Act2p partially overlaps that of conventional actin, indicatin g that it has a cytoskeletal function. In this study, both immunofluor escence localization and cellular fractionation of different epitope-t agged versions of Act2p also reveal an association with the nucleus, s uggesting an independent nuclear function for Act2p, Analysis of act2- 1 by electron microscopy, 30 min after a shift to tile restrictive tem perature (37 degrees C), reveals a striking aberration in NPC morpholo gy; NPCs appear as abnormal densities on either side of, rather than s panning, the nuclear envelope. Immunoelectron microscopy confirms that these densities contain XFXFG nucleoporins. act2-1 is synthetically l ethal in combination with a deletion in the XFXFG nucleoporin gene, NU P1, or a mutation in the nuclear localization sequence receptor gene, SRP1. Act2p and Srp1p co-immunoprecipitate, suggesting flat the protei ns exist in a complex, Together our data argue that Act2p plays an imp ortant role in NPC structure and function.