A yeast cell becomes committed to the cell division cycle only if it grows to a critical size and reaches a critical rate of protein synthesis. The coordination between growth and division takes place at a control step during the G1 phase of the cell cycle called Start. It relies on the G1-specific cyclins encoded by CLN1, 2 and 3, which trigger Start through the activation of the Cdc28 protein kinase. In fact, the Cln cyclins are rate-limiting for Start execution and depend on growth. Here we report that the cyclic AMP signal pathway modulates the dependency of Cln cyclins on growth. In particular, more growth is required to trigger Start because CLN1 and CLN2 are repressed by the cAMP signal, thus explaining the previously observed cAMP-dependent increase of the critical size and critical rate of protein synthesis. Cln3 is not inhibited by the cAMP pathway and counteracts this mechanism by partially mediating the growth-dependent expression of other G1 cyclins.
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.Throughout the eukaryotic kingdom cyclic AMP (cAMP)-dependent protein kinases (PKAs) play important and diverse roles in signal transduction (for reviews, see references 2, 7, and 28 and references therein). Structurally, PKAs are conserved, consisting of two catalytic subunits that bind, in their inactive configuration, to a regulatory subunit homodimer. Binding of cAMP to the regulatory subunit results in dissociation, and thereby activation, of the catalytic subunits (7, 28). The multitude of intracellular PKA substrates and their different subcellular distribution raises important questions about the specificity, timing, and substrate targeting of PKA-mediated signaling. One regulatory level to ensure proper signal transduction is specific targeting of signaling components to subcellular compartments. In multicellular eukaryotes A-kinase anchor proteins (AKAPs) have been identified that target type I or type II (RI or RII) PKA-regulatory subunits to their effector substrates localized in various subcellular compartments (for recent reviews, see references 5 and 6 and references therein). AKAPs possess a site for constitutive avid binding of RI or RII and a targeting domain that complexes with subcellular structures. Directing PKA to specific microenvironments facilitates phosphorylation of colocalized effector molecules.In contrast to cells from multicellular organisms, yeast...
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.
With the appropriate filter sets of both Bryte-HS (Bio-Rad, Hercules, CA) and FACScan (Becton Dickinson, San Jose, CA) flow cytometers, the measurement of separated green (SYBR Green I), orange-red (propidium iodide), and far red (RPE-Cy5) fluorescence was possible, allowing the enumeration of viable immunodetected bacteria. The entire protocol is completed in less than 3 h, offering numerous possibilities for rapid and precise analyses in sanitary, industrial, and environmental microbiology.
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