The vacuolar phosphatases purple acid phosphatase 26 and haloacid dehalogenase IIA2.1 hydrolyze 5′-, 3′-, and 2′-nucleotides derived from RNA degradation

The vacuole is an important site for RNA degradation. Autophagy delivers RNA to the vacuole, where the vacuolar T2 RNase ribonuclease 2 (RNS2) plays a major role in RNA catabolism. The presumed products of RNS2 activity are 3'-nucleoside monophosphates (3'-NMPs). Vacuolar phosphatases that carry out 3'-NMP hydrolysis are required to metabolize 3'-NMPs, but the specific players remain unknown. Using a mutant of RNS2 and mutants of the autophagy-related genes 5 and 9 (atg5 and atg9), we confirmed that 3'-NMPs are products of vacuolar RNS2-mediated RNA degradation in Arabidopsis (Arabidopsis thaliana). Moreover, we identified purple acid phosphatase 26 (PAP26) and haloacid dehalogenase IIA2.1 (HIIA2.1) as vacuolar 3'-NMP phosphatases. Based on phylogenetic analysis, we propose systematic nomenclature for HADIIA enzymes, some of which were previously named vegetative storage proteins, which we critically discuss. PAP26 and HIIA2.1 differ in their NMP specificity and activity in vitro. However, hiia2.1 pap26 double mutant plants, but generally not the respective single mutants, accumulate 3'-NMPs in addition to 5'-NMPs and, surprisingly, also 2'-NMPs. These findings suggest that PAP26 and HIIA2.1 have overlapping NMP substrate spectra in vivo. Excess 3'- and 2'-NMPs accumulate in plants exposed to a prolonged night, presumably because carbon limitation enhances autophagy-mediated vacuolar RNA degradation. We conclude that vacuolar RNA catabolism releases 3'-NMPs and 2'-NMPs through RNS2 and other RNases that also generate 5'-NMPs. PAP26 and HIIA2.1 are required to dephosphorylate these NMPs, so that they can enter general nucleotide metabolism outside the vacuole.