THE VACUOLAR TARGETING SIGNAL OF THE 2S ALBUMIN FROM BRAZIL NUT RESIDES AT THE C-TERMINUS AND INVOLVES THE C-TERMINAL PROPEPTIDE AS AN ESSENTIAL ELEMENT

Genetic constructs in which different N- and C-terminal segments of Br azil nut (Bertholletia excelsa H.B.K.) 2S albumin were fused to secret ory yeast invertase were transformed into tobacco (Nicotiana tabacum) plants to investigate the vacuolar targeting signal of the 2S albumin. None of the N-terminal segments, including the complete precursor con taining all propeptides, was able to direct the invertase to the vacuo les. However, a short C-terminal segment comprising the last 20 amino acids of the precursor was sufficient for efficient targeting of yeast invertase to the vacuoles of the transformed tobacco plants. Further analyses showed that peptides of 16 and 13 amino acids of the C-termin al segment were still sufficient, although they had slightly lower eff iciency. When segments of 9 amino acids or shorter were analyzed, a de crease to approximately 30% was observed. These segments included the C-terminal propeptide of four amino acids (Ile-Ala-Cly-Phe). When the 2S albumin was expressed in tobacco, it was also localized to the vacu oles of mesophyll cells. If the C-terminal propeptide was deleted from the 2S albumin precursor, all of this truncated 2S albumin was secret ed from the tobacco cells. These results indicate that the C-terminal propeptide is necessary but not sufficient for vacuolar targeting. In addition, an adjacent segment of at least 12 amino acids of the mature protein is needed to form the complete signal for efficient targeting .