ABSTRACT
Among 29 predicted Arabidopsis purple acid phosphatases (PAPs), AtPAP26 functions as the principle extracellular and intracellular PAP isozyme that is upregulated to recycle and scavenge Pi during Pi-deprivation or leaf senescence. Our companion paper documented the copurification of a secreted, high-mannose AtPAP26-S2 glycoform with AtGAL1 (At1g78850), a Pi starvation-inducible (PSI), and Galanthus nivalis agglutinin-related (mannose-binding) and apple domain lectin. This study tests the hypothesis that AtGAL1 binds AtPAP26-S2 to modify its enzymatic properties. Far-western immunodot blotting established that AtGAL1 readily associates with AtPAP26-S2 but not the low mannose AtPAP26-S1 glycoform nor other secreted PSI PAPs (i.e., AtPAP12 or AtPAP25). Analytical gel filtration indicated that 55-kDa AtGAL1 and AtPAP26-S2 polypeptides associate to form a 112-kDa heterodimer. Microscopic imaging of transiently expressed, fluorescent protein-tagged AtGAL1, and associated bimolecular fluorescence complementation assays demonstrated that (a) like AtPAP26, AtGAL1 also localizes to lytic vacuoles of Pi-deprived Arabidopsis and (b) both proteins interact in vivo. AtGAL1 preincubation significantly enhanced the acid phosphatase activity and thermal stability of AtPAP26-S2 but not AtPAP26-S1. We hypothesize that AtGAL1 plays an important role during Pi deprivation through its interaction with mannose-rich glycans of AtPAP26-S2 and consequent positive impact on AtPAP26-S2 activity and stability.
