Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles.

Katalin Petrényi,Joaquin Ariño,Ferenc Erdődi,Viktor Dombrádi,Cristina Molero,Zoltán Kónya

doi:10.1371/journal.pone.0160965

Katalin Petrényi, Joaquin Ariño + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0160965

Copy DOI

Abstract

Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a moonlighting protein.

Highlights

The Saccharomyces cerevisiae SIS2/HAL3 gene was discovered as a suppressor of the sit4 mutation [1] and a regulator of salt tolerance [2]
From the structural similarities between the Hal3 family members in S. cerevisiae and C. albicans we assumed that CaHal3 and CaCab3 will bind to the CaPpz1 phosphatase just as their budding yeast orthologs do [10]
Our present study was initiated on the basis of structural predictions suggesting a functional similarity between the orthologous C. albicans proteins, CaHal3 and CaCab3, and their S. cerevisiae counterparts ScHal3/ScVhs3 and ScCab3 (Fig 1)

Summary

Introduction

The Saccharomyces cerevisiae SIS2/HAL3 gene was discovered as a suppressor of the sit mutation [1] and a regulator of salt tolerance [2]. Subsequent work identified the gene VHS3 (a HAL3 paralog that arose from the whole genome duplication) encoding a second inhibitory subunit of Ppz1 [8] Both ScHal and ScVhs bind to the catalytic, C-terminal domain of Ppz1 [3,8] with a 1:1 stoichiometry [9]. While Ppz enzymes are found only in fungal species, orthologs of HAL3 were identified both in prokaryotic and eukaryotic organisms This ubiquitous distribution was explained by the fact that ScHal and ScVhs are moonlighting proteins that, in S. cerevisiae, constitute two subunits of a heterotrimeric phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme [10]. PPCDC catalyzes a key decarboxylation step in CoA biosynthesis Such heterotrimer would be composed from a constant ScCab (a ScHal and ScVhs paralog) subunit and two ScHal, two ScVhs or one of each (Fig 1). ScCab has some affinity for ScPpz, but cannot inhibit its phosphatase activity either in vitro or in vivo [10]

Methods

Results

Conclusion