Nucleocytoplasmic distribution of budding yeast protein kinase A regulatory subunit Bcy1 requires Zds1 and is regulated by Yak1-dependent phosphorylation of its targeting domain.

In Saccharomyces cerevisiae the subcellular distribution of Bcy1 is carbon source dependent. In glucose-grown cells, Bcy1 is almost exclusively nuclear, while it appears more evenly distributed between nucleus and cytoplasm in carbon source-derepressed cells. Here we show that phosphorylation of its N-terminal domain directs Bcy1 to the cytoplasm. Biochemical fractionation revealed that the cytoplasmic fraction contains mostly phosphorylated Bcy1, whereas unmodified Bcy1 is predominantly present in the nuclear fraction. Site-directed mutagenesis of two clusters (I and II) of serines near the N terminus to alanine resulted in an enhanced nuclear accumulation of Bcy1 in ethanol-grown cells. In contrast, substitutions to Asp led to a dramatic increase of cytoplasmic localization in glucose-grown cells. Bcy1 modification was found to be dependent on Yak1 kinase and, consequently, in ethanol-grown yak1 cells the Bcy1 remained nuclear. A two-hybrid screen aimed to isolate genes encoding proteins that interact with the Bcy1 N-terminal domain identified Zds1. In ethanol-grown zds1 cells, cytoplasmic localization of Bcy1 was largely absent, while overexpression of ZDS1 led to increased cytoplasmic Bcy1 localization. Zds1 does not regulate Bcy1 modification since this was found to be unaffected in zds1 cells. However, in zds1 cells cluster II-mediated, but not cluster I-mediated, cytoplasmic localization of Bcy1 was found to be absent. Altogether, these results suggest that Zds1-mediated cytoplasmic localization of Bcy1 is regulated by carbon source-dependent phosphorylation of cluster II serines, while cluster I acts in a Zds1-independent manner.

Nutritional control of nucleocytoplasmic localization of cAMP-dependent protein kinase catalytic and regulatory subunits in Saccharomyces cerevisiae.

In budding yeast, cAMP-dependent protein kinase (PKA) plays a central role in the nutritional control of metabolism, cell cycle, and transcription. This study shows that both the regulatory subunit Bcy1p and the catalytic subunit Tpk1p associated with it are predominantly localized in the nucleus of rapidly growing cells. Activation of nuclear PKA by cAMP leads to fast entry of a significant part of Tpk1p into the cytoplasm, while the regulatory subunit remains nuclear. In contrast to rapidly proliferating cells, both Bcy1p and Tpk1p are distributed over nucleus and cytoplasm in cells growing on a nonfermentable carbon source or in stationary phase cells. These results demonstrate that at least two different mechanisms determine the subcellular localization of PKA; cAMP controls the localization of Tpk1p, and the carbon source determines that of Bcy1p. The N-terminal domain of Bcy1p serves to target it properly during logarithmic and stationary phase. Studies with Bcy1p mutant versions unable to concentrate in the nucleus revealed that cells producing them are less viable in stationary phase than wild type cells, display delayed reproliferation following transfer to fresh growth medium, and, as diploids, exhibit reduced efficiency of sporulation.

Chromosome separation and exit from mitosis in budding yeast: dependence on growth revealed by cAMP-mediated inhibition.

Cell cycle progression of somatic cells depends on net mass accumulation. In Saccharomyces cerevisiae the cAMP-dependent kinases (PKAs) promote cytoplasmic growth and modulate the growth-regulated mechanism triggering the begin of DNA synthesis. By altering the cAMP signal in budding yeast cells we show here that mitotic events can also depend on growth. In fact, the hyperactivation of PKAs permanently inhibited both anaphase and exit from mitosis when cell growth was repressed. In S. cerevisiae the anaphase promoting complex (APC) triggers entry into anaphase by mediating the degradation of Pds1p. The cAMP pathway activation was lethal together with a partial impairment of the Cdc16p APC subunit, causing a preanaphase arrest, and conversely low PKA activity suppressed the lethality of cdc16-1 cells. Deregulated PKAs partially prevented the decrease of Pds1p intracellular levels concomitantly with the anaphase inhibition, and the PKA-dependent preanaphase arrest could be suppressed in pds1(-) cells. Thus, the cAMP pathway and APC functionally interact in S. cerevisiae and Pds1p is required for the cAMP-mediated inhibition of chromosome separation. Exit from mitosis requires APC, Cdc15p, and the polo-like Cdc5p kinase. PKA hyperactivation and a cdc15 mutation were synthetically lethal and brought to a telophase arrest. Finally, a low cAMP signal allowed cell division at a small cell size and suppressed the lethality of cdc15-2 or cdc5-1 cells. We propose that mitosis progression and the M/G1 phase transition specifically depend on cell growth through a mechanism modulated by PKAs and interacting with the APC/CDC15/CDC5 mitotic system. A possible functional antagonism between PKAs and the mitosis promoting factor is also discussed.