Abstract
Protein phosphatase Z is a “novel type” fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon) software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0) that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly suggested a role for CaPpz1 in biofilm formation, was confirmed experimentally. Thus our unbiased proteomic approach lead to the discovery of a novel function for this phosphatase in C. albicans.
Highlights
Protein phosphatase Z (PPZ) is a novel member of the Ser/Thr specific phosphoprotein phosphatase family that was identified by molecular cloning [1]
In this study we investigated the functional consequences of the deletion of the CaPPZ1 gene in C. albicans by using the combination of phosphoprotein and general protein staining of control QMY23 (Ctrl) and deletion mutant cappz1 (Mut) strains
In comparison with S. cerevisiae, where the EF1Bα/Efb1 translational elongation factor was reported to be a Ppz1 substrate/target [13], we found that the amount of the C. albicans ortholog of the Efb1 was significantly reduced in the phosphatase mutant, but the phosphorylated form of this protein we were not able to detect by the Pro-Q Diamond staining (Table 1)
Summary
Protein phosphatase Z (PPZ) is a novel member of the Ser/Thr specific phosphoprotein phosphatase family that was identified by molecular cloning [1]. This phosphatase has a distinctive phylogenetic distribution as it is specific for fungi. Due to several unique structural features—including a specific C-terminal helix—a number of point mutations in important binding sites, and the presence of its N-terminal tail [8] CaPpz exhibits distinct regulation and has different physiological functions in comparison to the classical PP1c termed Glc in C. albicans
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