Elucidation and application of a novel mechanism regulating vacuolar localization of plant proteins

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Aureusidin synthase, a polyphenol oxidase (PPO), specifically catalyzes the oxidative formation of aurones from chalcones, which are plant flavonoids, and is responsible for the yellow coloration of snapdragon flowers. All known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place in cytoplasm (or on the cytoplasmic surface of ER). However, primary structural characteristics and some molecular properties of aureusidin synthase contradict the enzyme's localization in plastids and cytoplasm. In this study, the subcellular localization of this enzyme in petal cells of the yellow snapdragon was investigated. Sucrose-density gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme was localized within vacuole lumen. We also found that the necessary information for the vacuolar targeting of the PPO was encoded within the 53-residue N-terminal sequence (NTPP), but not in C-terminal sequence of the precursor. The NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera fused with a dominant negative mutant of Sari GTPase of Arabidopsis or that with mRFP-fused Glogi marker (H^+-translocating inorganic pyrophosphatase of Arabidopsis). We identified a sequence-specific vacuolar sorting determinant in the NTPP of the precursor. This provides the first example of the biosynthesis of a flavonoid skeleton in vacuoles. This metabolic compartmentation should serve as a strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower.