Regulation of the Yeast Ace2 Transcription Factor during the Cell Cycle*

Abstract

Previous studies have revealed many parallels in the cell cycle regulation of the Ace2 and Swi5 transcription factors. Although both proteins begin entry into the nucleus near the start of mitosis, here we show that Ace2 accumulates in the nucleus and binds DNA about 10 min later in the cell cycle than Swi5. We used chimeric fusions to identify the N-terminal region of Ace2 as responsible for the delay, and this same region of Ace2 was required for interaction with Cbk1, a kinase necessary for both transcriptional activation by Ace2 and asymmetric distribution of Ace2. Ace2 and Swi5 also showed differences in prevalence during the cell cycle. Swi5 is apparently degraded soon after nuclear entry, whereas constant Ace2 levels throughout the cell cycle suggest Ace2 is exported from the nucleus. Our work suggests that the precise timing of Ace2 accumulation in the nucleus involves both a nuclear export sequence and a nuclear localization signal, whose activities are regulated by phosphorylation.Ace2 and Swi5 are yeast transcription factors with a number of similarities. Previous work has shown that ACE2 and SWI5 are both transcribed during the G 2 portion of the cell cycle, and the Ace2 and Swi5 proteins accumulate in the nucleus at roughly the same time early in M phase (1-3). Additionally the zinc finger DNA-binding domains of the two proteins are nearly identical, and Ace2 and Swi5 recognize the same DNA sequences in vitro (3, 4). Nonetheless Ace2 and Swi5 activate transcription of different genes in vivo (3, 5). Ace2 activates expression of the CTS1 gene, whereas Swi5 does not. In contrast, Swi5 is required for HO expression, whereas Ace2 is unable to activate HO (3).The mechanisms controlling the cell cycle regulation of Ace2 and Swi5 have been investigated. The G 2 -specific transcription of the ACE2 and SWI5 genes is due to binding sites for the Mcm1-Fkh2-Ndd1 complex in both promoters (6 -10). A nuclear localization signal (NLS) 5 was identified in Swi5, and phosphorylation in the vicinity of the NLS by the Cdc28 cyclindependent kinase prevents nuclear accumulation (1). Swi5 is dephosphorylated at anaphase by the Cdc14 phosphatase (11). Importantly mutation of the three phosphorylated serine residues in Swi5 responsible for down-regulation of the NLS results in constitutive nuclear localization of Swi5. The amino acids in Swi5 that are phosphorylated to regulate nuclear localization are conserved in Ace2 (3), and mutation of these residues results in constitutive nuclear localization (12).Ace2 interacts with the Crm1 nuclear export receptor and contains a nuclear export sequence (NES) (13). A GFP-Ace2 fusion is cytoplasmic, but it is nuclear in the presence of the leptomycin B nuclear export inhibitor (13) or in cells with a mutation in the CRM1 nuclear export protein (14). Importantly a GFP-Swi5 fusion does not respond to leptomycin B. Leucine residues are an essential part of all previously characterized NESs, but mutagenesis of this putative NES region in Ace2 shows that leucines are not required for NES ...

Mitotic Exit Control of the Saccharomyces cerevisiae Ndr/LATS Kinase Cbk1 Regulates Daughter Cell Separation after Cytokinesis

“…5A). This was unsurprising because cdc15-2 cells arrest prior to large-scale release of Cdc14, which mediates dephosphorylation of the CDK sites in wildtype Ace2 (43,55). By examining localization of Ace2-AAA-GFP, we found that cdc15-2 cells grown at permissive temperature exhibit the same Ace2-AAA-GFP nuclear localization pattern as wild-type cells (data not shown).…”

“…Differences between the behavior of Ace2 and the closely related transcription factor Swi5 indicate that Ace2's regulatory system does in fact generate delayed output. In mitotically synchronized cells, Swi5 accumulates in nuclei significantly before Ace2 (55). Like Ace2, Swi5 is blocked from entering nuclei in mitosis by CDK phosphorylation (47), and Cdc14 reverses this (67).…”

“…Therefore, to assess Cbk1's regulation of Ace2 more directly, we eliminated mitotic CDK control of Ace2's nuclear entry by changing phospho-acceptor residues in three key CDK sites to alanine. This allele, referred to as ace2-AAA, eliminates mitotic CDK inhibition of Ace2 nuclear import (50,55). With these inhibitory CDK sites removed, nuclear accumulation and transcriptional activity of Ace2 are regulated only by Cbk1's inactivation of the Ace2 NES (42).…”

“…Correct timing of this division event is partly achieved through control of transcription: the enzymes that degrade the septum are produced in a brief pulse of gene transcription as cells pass from mitosis to G 1 phase. The activation of these genes is driven by the transcription factor Ace2, which localizes strongly to the daughter nucleus immediately before completion of actomyosin ring contraction (8,16,18,19,42,50,55). Ace2 is regulated by sequential inhibition and activation.…”

New Insight Into the Role of the Cdc34 Ubiquitin-Conjugating Enzyme in Cell Cycle Regulation via Ace2 and Sic1

“…Western immunoblot analysis was performed by standard methods. Antibodies were used at the following dilutions: affinity-purified anti-Cdc34 (1:10,000), affinity-purified anti-Sic1 (1:1000), Ace2 antisera (1:5000) (Sbia et al 2008), and anti-TAP (1:2000) (Open Biosystems). Primary antibody was detected with an HRPconjugated goat anti-rabbit secondary antibody at a 1:10,000 dilution (Santa Cruz Biotechnology).…”