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* (RiVTC4^183–474) 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.

摘要

丛枝菌根 (AM) 真菌与陆地植物形成共生关系，并向宿主提供包括磷在内的土壤矿物质。AM 真菌在其菌丝体中积累多磷酸盐 (polyP)，这是一种线性磷酸盐聚合物，其在磷储存和转运中起作用。在出芽酵母酿酒酵母中，已经证明液泡转运蛋白伴侣 4 (VTC4) 蛋白是 VTC 复合物的一个亚基，负责 polyP 的合成。在这里，我们通过 Blast 分析对八种 AM 真菌基因组中的 VTC 蛋白进行了全面调查，并表征了不规则根霉 VTC4 的生化特性。AM 真菌物种的基因组编码 VTC1、VTC2 和 VTC4。重组蛋白 RiVTC4* (RiVTC4 ^183–474) 含有催化隧道结构域在大肠杆菌细胞中表达并纯化。RiVTC4* 能够使用 ATP 作为底物催化 polyP 聚合。焦磷酸盐增强 polyP 聚合活性 > 10 倍。RiVTC4* 在中性 pH 条件下表现出最大活性，并且需要二价金属离子，优选 Mn ²⁺。在高浓度 ADP 存在下，RiVTC4* 发生逆反应（从 polyP 再生 ATP）。在 0.2-5 mM ADP 的范围内，在 ATP/ADP 比率小于 2-5 时观察到通过逆反应进行的 polyP 解聚。这些结果表明，AM 真菌 VTC4 不仅可以合成 polyP，还可以从 polyP 和 ADP 中再生 ATP，这对调节 AM 真菌中的 polyP 和 ATP 水平具有潜在意义。