SummaryIn the yeast Saccharomyces cerevisiae , PKA and Sch9 exert similar physiological roles in response to nutrient availability. However, their functional redundancy complicates to distinguish properly the target genes for both kinases. In this article, we analysed different phenotypic read-outs. The data unequivocally showed that both kinases act through separate signalling cascades. In addition, genome-wide expression analysis under conditions and with strains in which either PKA and/or Sch9 signalling was specifically affected, demonstrated that both kinases synergistically or oppositely regulate given gene targets. Unlike PKA, which negatively regulates stress-responsive element (STRE)-and post-diauxic shift (PDS)-driven gene expression, Sch9 appears to exert additional positive control on the Rim15-effector Gis1 to regulate PDS-driven gene expression. The data presented are consistent with a cyclic AMP (cAMP)-gating phenomenon recognized in higher eukaryotes consisting of a main gatekeeper, the protein kinase PKA, switching on or off the activities and signals transmitted through primary pathways such as, in case of yeast, the Sch9-controlled signalling route. This mechanism allows fine-tuning various nutritional responses in yeast cells, allowing them to adapt metabolism and growth appropriately.

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.

In this work, uniform manifold approximation and projection (UMAP) is applied for nonlinear dimensionality reduction and visualization of mass spectrometry imaging (MSI) data. We evaluate the performance of the UMAP algorithm on MSI data sets acquired in mouse pancreas and human lymphoma samples and compare it to those of principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), and the Barnes−Hut (BH) approximation of t-SNE. Furthermore, we compare different distance metrics in (BH) t-SNE and UMAP and propose the use of spatial autocorrelation as a means of comparing the resulting low-dimensional embeddings. The results indicate that UMAP is competitive with t-SNE in terms of visualization and is well-suited for the dimensionality reduction of large (>100 000 pixels) MSI data sets. With an almost fourfold decrease in runtime, it is more scalable in comparison with the current state-of-the-art: t-SNE or the Barnes−Hut approximation of t-SNE. In what seems to be the first application of UMAP to MSI data, we assess the value of applying alternative distance metrics, such as the correlation, cosine, and the Chebyshev metric, in contrast to the traditionally used Euclidean distance metric. Furthermore, we propose "histomatch" as an additional custom distance metric for the analysis of MSI data.

A yeast model was generated to study the mechanisms and phenotypical repercussions of expression of α‐synuclein as well as the coexpression of protein tau. The data show that aggregation of α‐synuclein is a nucleation–elongation process initiated at the plasma membrane. Aggregation is consistently enhanced by dimethyl sulfoxide, which is known to increase the level of phospholipids and membranes in yeast cells. Aggregation of α‐synuclein was also triggered by treatment of the yeast cells with ferrous ions, which are known to increase oxidative stress. In addition, data are presented in support of the hypothesis that degradation of α‐synuclein occurs via autophagy and proteasomes and that aggregation of α‐synuclein disturbs endocytosis. Reminiscent of observations in double‐transgenic mice, coexpression of α‐synuclein and protein tau in yeast cells is synergistically toxic, as exemplified by inhibition of proliferation. Taken together, the data show that these yeast models recapitulate major aspects of α‐synuclein aggregation and cytotoxicity, and offer great potential for defining the underlying mechanisms of toxicity and synergistic actions of α‐synuclein and protein tau.

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cAMP-dependent protein kinases (PKA) are ubiquitous signalling molecules that mediate many extracellular signals in eukaryotes from yeast to men. Directing PKA to its substrates is an important level of control to ensure specificity of cAMP-mediated signal transduction. Unlike in yeast and fungi, in mammalian cells a relatively sophisticated insight has been obtained in the controls of PKA localisation and in fact has set the stage for future research on PKA targeting in unicellular eukaryotes. In this review, we present an integrated overview on molecular mechanisms of PKA regulatory and catalytic subunit localisation in both yeast and multicellular organisms; and we focus in more detail on recent advances of PKA localisation in the unicellular eukaryote Saccharomyces cerevisiae.

We have developed a yeast-based model recapitulating neurotoxicity of alpha-synuclein fibrilization. This model recognized metal ions, known risk factors of alpha-synucleinopathy, as stimulators of alpha-synuclein aggregation and cytotoxicity. Elimination of Yca1 caspase activity augmented both cytotoxicity and inclusion body formation, suggesting the involvement of apoptotic pathway components in toxic alpha-synuclein amyloidogenesis. Deletion of hydrophobic amino acids at positions 66-74 in alpha-synuclein reduced its cytotoxicity but, remarkably, did not lower the levels of insoluble alpha-synuclein, indicating that noxious alpha-synuclein species are different from insoluble aggregates. A compound screen aimed at finding molecules with therapeutic potential identified flavonoids with strong activity to restrain alpha-synuclein toxicity. Subsequent structure-activity analysis elucidated that these acted by virtue of anti-oxidant and metal-chelating activities. In conclusion, this yeast-cell model as presented allows not only fundamental studies related to mechanisms of alpha-synuclein-instigated cellular degeneration, but is also a valid high-throughput identification tool for novel neuroprotective agents.