Rice plastidial N-glycosylated nucleotide pyrophosphatase/phosphodiesterase is transported from the ER-Golgi to the chloroplast through the secretory pathway

A nucleotide pyrophosphatase/ phosphodiesterase ( NPP) activity that catalyzes the hydrolytic breakdown of ADP- glucose ( ADPG) has been shown to occur in the plastidial compartment of both mono- and dicotyledonous plants. To learn more about this enzyme, we purified two NPPs from rice ( Oryza sativa) and barley ( Hordeum vulgare) seedlings. Both enzymes are glycosylated, since they bind to concanavalin A, stain with periodic acid - Schiff reagent, and are digested by Endo- H. A complete rice NPP cDNA, designated as NPP1, was isolated, characterized, and overexpressed in transgenic plants displaying high ADPG hydrolytic activity. Databank searches revealed that NPP1 belongs to a functionally divergent group of plant nucleotide hydrolases. NPP1 contains numerous N- glycosylation sites and a cleavable hydrophobic signal sequence that does not match with the N- terminal part of the mature protein. Both immunocytochemical analyses and confocal fluorescence microscopy of rice cells expressing NPP1 fused with green fluorescent protein ( GFP) revealed that NPP1- GFP occurs in the plastidial compartment. Brefeldin A treatment of NPP1- GFP - expressing cells prevented NPP1- GFP accumulation in the chloroplasts. Endo- H digestibility studies revealed that both NPP1 and NPP1- GFP in the chloroplast are glycosylated. Collectively, these data demonstrate the trafficking of glycosylated proteins from the endoplasmic reticulum - Golgi system to the chloroplast in higher plants.