Ser/Thr protein phosphatases in fungi: structure, regulation and function – Reversible phospho-dephosphorylation of proteins is a major mechanism for the control of cellular functions. By large, Ser and Thr are the most frequently residues phosphorylated in eukaryotes. Removal of phosphate from these amino acids is catalyzed by a large family of well-conserved enzymes, collectively called Ser/Thr protein phosphatases. The activity of these enzymes has an enormous impact on cellular functioning. In this work we present the members of this family in S. cerevisiae and other fungal species, and review the most recent findings concerning their regulation and the roles they play in the most diverse aspects of cell biology.

Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1–45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance.

The alteration of the fine-tuned balance of phospho/dephosphorylation reactions in the cell often results in functional disturbance. In the yeast Saccharomyces cerevisiae, the overexpression of Ser/Thr phosphatase Ppz1 drastically blocks cell proliferation, with a profound change in the transcriptomic and phosphoproteomic profiles. While the deleterious effect on growth likely derives from the alteration of multiple targets, the precise mechanisms are still obscure. Ppz1 is a negative effector of potassium influx. However, we show that the toxic effect of Ppz1 overexpression is unrelated to the Trk1/2 high-affinity potassium importers. Cells overexpressing Ppz1 exhibit decreased K⁺ content, increased cytosolic acidification, and fail to properly acidify the medium. These effects, as well as the growth defect, are counteracted by the deletion of NHA1 gene, which encodes a plasma membrane Na⁺, K⁺/H⁺ antiporter. The beneficial effect of a lack of Nha1 on the growth vanishes as the pH of the medium approaches neutrality, is not eliminated by the expression of two non-functional Nha1 variants (D145N or D177N), and is exacerbated by a hyperactive Nha1 version (S481A). All our results show that high levels of Ppz1 overactivate Nha1, leading to an excessive entry of H⁺ and efflux of K⁺, which is detrimental for growth.

S.cerevisiaeHal3 (ScHal3) is a moonlighting protein that, is in its monomeric state, regulates the Ser/Thr protein phosphatase Ppz1, but also joins ScCab3 (and in some instances the Hal3 paralog Vhs3) to form an unusual heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. PPCDC is required for CoA biosynthesis and in most eukaryotes is a homotrimeric complex with three identical catalytic sites at the trimer interfaces. However, inS.cerevisiaethe heterotrimeric arrangement results in a single functional catalytic center. Importantly, the specific structural determinants that direct Hal3’s oligomeric state and those required for Ppz1 inhibition remain largely unknown. We mutagenized residues in the predicted hydrophobic core of ScHal3 (L403–L405) and the plantArabidopsis thalianaHal3 (AtHal3, G115–L117) oligomers and characterized their properties as PPCDC components and, for ScHal3, also as Ppz1 inhibitor. We found that in AtHal3 these changes do not affect trimerization or PPCDC function. Similarly, mutation of ScHal3 L403 has no effect. In contrast, ScHal3 L405E fails to form homotrimers, but retains the capacity to bind Cab3—explaining its ability to rescue ahal3 vhs3synthetically lethal mutation. Remarkably, the L405E mutation decreases Hal3’s ability to interact with and to inhibit Ppz1, confirming the importance of the oligomer/monomer equilibrium in Hal3’s Ppz1 regulating function.