Berberine bridge enzyme, a key branch-point enzyme in benzylisoquinoline alkaloid biosynthesis, contains a vacuolar sorting determinant

In opium poppy (Papaver somniferum L.), (S)-reticuline is the last common i ntermediate in sanguinarine and morphine biosynthesis. Sanguinarine accumul ates in the vacuole of cultured opium poppy cells in response to treatment with fungal elicitors. The first committed step in sanguinarine biosynthesi s is catalyzed by the berberine bridge enzyme (BBE), which converts (S)-ret iculine to (S)-scoulerine. An N-terminal signal peptide and novel vacuolar sorting determinant were identified and characterized in BBE. In vitro tran slation of BBE mRNA in the presence of canine pancreatic microsomes produce d a glycosylated, proteolysis-resistant protein, confirming the existence o f a signal peptide. Transcripts encoding a BBE N-terminal deletion series f used to beta -glucuronidase or green fluorescent protein (GFP) were also tr anslated in the presence of canine microsomes, and introduced into cultured opium poppy cells via microprojectile bombardment. The signal peptide was restricted to the first 25 amino acids and shown to initially target BBE to the endoplasmic reticulum. Fusion of 50 N-terminal residues from BBE to GF P resulted in the localization of the reporter to the vacuole. GFP was also sorted to the vacuole when fused to a heterologous N-terminal signal pepti de followed by BBE amino acids 26-50. The BBE vacuolar sorting determinant was further localized between residues 26 and 41 by deletion analysis. The final subcellular destination of BBE is consistent with the vacuolar seques tration of sanguinarine. However, the vacuolar pH is below the functional r ange for BBE, suggesting that the enzyme is active only prior to its entry into the vacuole.