Polyphosphate polymerizing and depolymerizing activity of VTC4 protein in an arbuscular mycorrhizal fungus

Abstract

ABSTRACT Arbuscular mycorrhizal (AM) fungi form symbiotic associations with land plants and supply soil minerals including phosphorus to their hosts. AM fungi accumulate polyphosphate (polyP), a linear phosphate polymer, in their mycelia, which functions in phosphorus storage and translocation. In the budding yeast Saccharomyces cerevisiae, it has been demonstrated that the vacuolar transporter chaperone 4 (VTC4) protein, a subunit of the VTC complex, is responsible for polyP synthesis. Here, we conducted a comprehensive survey of VTC proteins in eight AM fungal genomes by Blast analysis and characterized the biochemical properties of the Rhizophagus irregularis VTC4. The genomes of AM fungal species encode VTC1, VTC2, and VTC4. The recombinant protein RiVTC4* (RiVTC4183–474) containing the catalytic tunnel domain was expressed in E. coli cells and purified. RiVTC4* is capable of catalyzing polyP polymerization using ATP as a substrate. Pyrophosphate enhanced polyP-polymerizing activity >10-fold. RiVTC4* exhibited maximum activity at neutral pH and required divalent metal ions, preferentially Mn2+. In the presence of high concentrations of ADP, the reverse reaction (the regeneration of ATP from polyP) by RiVTC4* occurred. In the range of 0.2–5 mM ADP, polyP depolymerization by the reverse reaction was observed at the ATP/ADP ratio of less than 2–5. These results suggest that AM fungal VTC4 not only synthesizes polyP but also regenerates ATP from polyP and ADP, which has potential implications for the modulation of polyP and ATP levels in AM fungi.