Functional Characterization of GmALA1, a Plasma Membrane-Localized P4-ATPase, and Its Interacting β-Subunit GmALIS2...

P4-ATPases maintain transbilayer lipid asymmetry, yet their functional roles in legume crops remain poorly understood. In the present study, GmALA1 a plasma membrane-localized P4-ATPase in soybean, was identified and characterized. Its physical interaction with the β-subunit GmALIS2 at the plasma membrane via biomolecular fluorescence complementation was confirmed. Heterologous expression in the P4-ATPase-deficient yeast strain ZHY709 demonstrated that GmALA1 fully complemented the cold-sensitive growth phenotype, while co-expression with GmALIS2 only partially restored growth, suggesting GmALIS2 may modulate rather than simply stimulate GmALA1 activity, though the mechanism remains unresolved. GmALA1 suppresses triacylglycerol accumulation while elevating lysophosphatidylethanolamine and lysophosphatidylcholine content in both wild-type and mutant yeast. These findings were consistent with GmALA1-driven remodeling of membrane lipid flux. In yeast and transgenic soybean hairy roots, GmALA1 alone or in combination with GmALIS2 differentially altered the internalization and tissue-specific distribution of multiple phospholipid classes, with the pattern of NBD-lipid accumulation differing depending on GmALIS2 co-expression and cellular context. GmALA1 expression was also associated with altered yeast sensitivity to divalent cations including Ca²⁺, Co²⁺, and Zn²⁺. Also, cellular cation accumulation in the P4-ATPase-deficient background was enhanced. However, whether this reflects a direct interaction between GmALA1 and cation homeostasis machinery remains to be established. These findings establish GmALA1 as a functionally active phospholipid flippase that coordinates transmembrane lipid redistribution in concert with GmALIS2. These findings advance our understanding of P4-ATPase biology in soybean and legume crops.