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

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.

蛋白磷酸酶Z（Protein phosphatase Z，Ppz）是一类真菌特异性酶，可调控细胞壁完整性、阳离子稳态与氧化应激应答。针对酿酒酵母（Saccharomyces cerevisiae）的研究表明，该酶可被Hal3、Vhs3兼职蛋白（moonlighting protein）抑制，这类蛋白与Cab3共同构成了必需的磷酸泛酰半胱氨酸脱羧酶（phosphopantothenoylcysteine decarboxylase，PPCDC）。在白色念珠菌（Candida albicans）中，CaPpz1还参与了该机会致病性人类病原菌的形态变化与感染过程。为阐明CaPpz1的调控网络，我们检索了白色念珠菌数据库，鉴定出两个基因；基于其酿酒酵母同源蛋白的结构特征，将它们分别命名为CaHal3与CaCab3。通过下拉实验（pull down assay）与磷酸酶活性测定，我们证实这两种细菌表达的重组蛋白均可结合并抑制CaPpz1及其C端催化结构域（CaPpz1-Cter），且二者的作用效率相当。CaPpz1-Cter的结合与抑制作用始终更为显著，提示全长蛋白中的N端结构域可对抑制作用产生保护效应。我们通过在酿酒酵母中过表达上述白色念珠菌蛋白，对其功能进行了验证。与预期相悖的是，仅CaCab3而非CaHal3可挽救由ScHal3介导的磷酸酶抑制相关表型特征，例如对氯化锂（LiCl）或潮霉素B（hygromycin B）的耐受性、对胞外钾离子浓度的需求，或是在丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）缺陷型slt2菌株背景下的生长能力。另一方面，这两种念珠菌蛋白均被证实为PPCDC的必需组成成分，且其功能与酿酒酵母同源蛋白一致：CaCab3与CaHal3的表达可分别挽救酿酒酵母cab3突变体与hal3 vhs3双突变体的表型。因此，CaHal3与CaCab3均保留了与PPCDC相关的功能，且在体外均具备抑制CaPpz1的潜力。仅有CaCab3在体内展现出磷酸酶调控潜力这一事实表明，在白色念珠菌中，CaCab3而非CaHal3发挥兼职蛋白的功能。