Abstract
Phosphate is the essential macronutrient required for the growth of all organisms. In Saccharomyces cerevisiae, phosphatases are up-regulated, and the level of lysophosphatidic acid (LPA) is drastically decreased under phosphate-starved conditions. The reduction in the LPA level is attributed to PHM8, a gene of unknown function. phm8Delta yeast showed a decreased LPA-hydrolyzing activity under phosphate-limiting conditions. Overexpression of PHM8 in yeast resulted in an increase in the LPA phosphatase activity in vivo. In vitro assays of the purified recombinant Phm8p revealed magnesium-dependent LPA phosphatase activity, with maximal activity at pH 6.5. The purified Phm8p did not hydrolyze any lipid phosphates other than LPA. In silico analysis suggest that Phm8p is a soluble protein with no transmembrane domain. Site-directed mutational studies revealed that aspartate residues in a DXDXT motif are important for the catalysis. These findings indicated that LPA plays a direct role in phosphate starvation. This is the first report of the identification and characterization of magnesium-dependent soluble LPA phosphatase.
Highlights
Phosphate is the most common functional group present in the metabolome
The decrease in lysophosphatidic acid (LPA) concentration in our study is attributed to PHM8, which is induced during phosphate starvation
To authenticate the involvement of PHM8 in dephosphorylation of LPA during phosphate starvation, we have extracted lipids from the phm8⌬ mutant yeast cells grown in complete synthetic medium and phosphate-deprived medium that were subjected to Electrospray Ionization Mass Spectrometry (ESI-MS) analyses (Fig. 3, A and B)
Summary
Materials—S. cerevisiae (BY4741) and PHM8 knock-out (phm⌬ mutant) in BY4741 strains were obtained from Euroscarf (Frankfurt, Germany). [32P]Orthophosphate (3000 sulfonic acid; ESI-MS, electrospray ionization-mass spectrometry; IPTG, isopropyl-1-thio--D-galactopyranoside.
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