Abstract
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.
Highlights
Saccharomyces cerevisiae Ppz1 is a type 1-related Ser/Thr protein phosphatase (692aminoacid residues) composed of a C-terminal catalytic domain and a long N-terminal extension of about 350 residues [1,2]
Ppz1 is regulated by two inhibitory subunits, Hal3 and Vhs3 [5,6,7], Hal3 being the more relevant inhibitor in vivo. These are moonlighting proteins since, in addition to their role in Ppz1 inhibition, they associate with Cab3, forming an unusual heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC) involved in the biosynthesis of coenzyme A [7,8,9]
Because Ppz1 has been recognized as a virulence factor in important human pathogenic fungi, such as Candida albicans [15] and Aspergillus fumigatus [16], as well as in the plant pathogen Colletotrichum gloeosporioides [17], and this protein is not present in animals or plants, it has been proposed as a possible antifungal target
Summary
Saccharomyces cerevisiae Ppz is a type 1-related Ser/Thr protein phosphatase (692aminoacid residues) composed of a C-terminal catalytic domain and a long N-terminal extension of about 350 residues [1,2]. Ppz plays a key role in monovalent cation homeostasis, and it was demonstrated long ago that the deletion of PPZ1 results in an increased salt tolerance [18] This effect has been ascribed to the role of Ppz in two different processes: (i) the inhibition of potassium uptake through the K+ high-affinity Trk and Trk transporters, and (ii) a repressive effect on the expression of the Ena Na+, K+-ATPase involved in the response to salt stress [18,19,20,21]. A reasonable hypothesis to explain Ppz toxicity was that high Ppz levels would block Trk1/2-mediated K+ uptake, a situation incompatible with cell proliferation under standard growth conditions Another transport system involved in the maintenance of monovalent cation homeostasis is the plasma-membrane Nha antiporter, constitutively produced in cells in low amounts [23]. We must conclude that the overexpression of Ppz does alter monovalent cation homeostasis in a way that involves the plasma membrane Na+, K+/H+ antiporter Nha
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