Protein Phosphatase Ppz1 Is Not Regulated by a Hal3-Like Protein in Plant Pathogen Ustilago maydis.

Chunyi Zhang,Antonio De La Torre,Joaquín Ariño,José Pérez-Martín

doi:10.3390/ijms20153817

Abstract

Ppz enzymes are type-1 related Ser/Thr protein phosphatases that are restricted to fungi. In S. cerevisiae and other fungi, Ppz1 is involved in cation homeostasis and is regulated by two structurally-related inhibitory subunits, Hal3 and Vhs3, with Hal3 being the most physiologically relevant. Remarkably, Hal3 and Vhs3 have moonlighting properties, as they participate in an atypical heterotrimeric phosphopantothenoyl cysteine decarboxylase (PPCDC), a key enzyme for Coenzyme A biosynthesis. Here we identify and functionally characterize Ppz1 phosphatase (UmPpz1) and its presumed regulatory subunit (UmHal3) in the plant pathogen fungus Ustilago maydis. UmPpz1 is not an essential protein in U. maydis and, although possibly related to the cell wall integrity pathway, is not involved in monovalent cation homeostasis. The expression of UmPpz1 in S. cerevisiae Ppz1-deficient cells partially mimics the functions of the endogenous enzyme. In contrast to what was found in C. albicans and A. fumigatus, UmPpz1 is not a virulence determinant. UmHal3, an unusually large protein, is the only functional PPCDC in U. maydis and, therefore, an essential protein. However, when overexpressed in U. maydis or S. cerevisiae, UmHal3 does not reproduce Ppz1-inhibitory phenotypes. Indeed, UmHal3 does not inhibit UmPpz1 in vitro (although ScHal3 does). Therefore, UmHal3 might not be a moonlighting protein.

Highlights

Ppz1 defines a subfamily of Ser/Thr protein phosphatases (PPases) which are structurally related to type-1 (PP1c) enzymes
The expression of UmPpz1 in S. cerevisiae shows that this phosphatase could partially replace endogenous Ppz1 in S. cerevisiae, suggesting that the Ustilago enzyme has the capacity to impact on specific intracellular targets of the Saccharomyces phosphatase
This motif is absent in the N-terminal region of CnPpz1, and expression of this enzyme in S. cerevisiae partially normalizes Li+ tolerance [24]

Summary

Introduction

Ppz defines a subfamily of Ser/Thr protein phosphatases (PPases) which are structurally related to type-1 (PP1c) enzymes. In contrast to the ubiquitous PP1c, Ppz phosphatases are restricted to fungi [1,2]. S. cerevisiae ppz mutants are hypertolerant to Na+ and Li+ cations and, likely due to the increased influx of potassium, they are sensitive to agents affecting cell wall remodeling, such as caffeine or calcofluor white [4,9,10]. High levels of Ppz are toxic in S. cerevisiae, resulting in slow growth and cell cycle blockage at the G1–S transition [9,11,12]. Hal is more relevant in vivo than Vhs and, in contrast to ppz strains, hal mutants are sensitive to Na+ and Li+, whereas its overexpression confers tolerance to high concentrations of these cations [13]

Methods

Results

Conclusion