A role for the divergent actin gene, ACT2, in nuclear pore structure and function

We have identified a temperature-sensitive allele of the yeast divergent actin gene ACT2, act2-1, which displays defects in nuclear pore complex (NPC) structure and nuclear import at the restrictive temperature. Although defective in nuclear import, act2-1 cells still selectively retain reporter proteins in the nucleus, and by indirect immunofluorescence the actin cytoskeleton appears normal. Previous studies in Acanthamoeba and Saccharomyces cerevisiae reported that the cellular location of Act2p partially overlaps that of conventional actin, indicating that it has a cytoskeletal function. In this study, both immunofluorescence localization and cellular fractionation of different epitope-tagged versions of Act2p also reveal an association with the nucleus, suggesting an independent nuclear function for Act2p. Analysis of act2-1 by electron microscopy, 30 min after a shift to the restrictive temperature (37 degrees C), reveals a striking aberration in NPC morphology; NPCs appear as abnormal densities on either side of, rather than spanning, the nuclear envelope. Immunoelectron microscopy confirms that these densities contain XFXFG nucleoporins. act2-1 is synthetically lethal in combination with a deletion in the XFXFG nucleoporin gene, NUP1, or a mutation in the nuclear localization sequence receptor gene, SRP1. Act2p and Srp1p co-immunoprecipitate, suggesting that the proteins exist in a complex. Together our data argue that Act2p plays an important role in NPC structure and function.