Identification of Novel Saccharomyces cerevisiaeProteins with Nuclear Export Activity: Cell Cycle-Regulated Transcription Factor Ace2p Shows Cell Cycle-Independent Nucleocytoplasmic Shuttling

Abstract

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiaeproteins not previously known to have nuclear export activity. These proteins are the 5′ RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G₁-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G₁ but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner.

ACKNOWLEDGMENTS

We thank Pam Silver for the gift of plasmids pPS808, pPS1372, and JH23 and Neal Sugawara and Yval Blat for advice on cell cycle arrest. We also thank Minoru Yoshida for generously providing LMB, Ed Dougherty for help with figures, and members of the Rosbash lab for stimulating discussions.

T.H.J. was supported by grants from the Carlsberg Foundation and partly from the Leo Nielsen and Løvens Kemiske Fabrik foundations. This work was supported by NIH grant GM 23549.