Molecular basis of post-translational modifications and targeting of barley lectin to the vacuoles in barley and in transgenic tobacco plants
1993
Schroeder, M.R. | Dombrowski, J.E. | Bednarek, S.Y. | Borkhsenious, O.N. | Raikhel, N.V.
Secretory proteins are sorted to the plant cell vacuoles or are retained within the secretory pathway by mechanisms which require specific targeting information contained within the structure of the protein. It has been demonstrated that secretory proteins lacking such information follow a default pathway and are secreted. We have previously shown, using a plant transgenic system, that the 15-amino acid carboxyl-terminal propeptide (CTPP) of probarley lectin (proBL) is necessary for the proper sorting of this protein to the plant vacuole. Mutant forms of the protein lacking the CTPP are secreted. To test the hypothesis that the CTPP is sufficient as a vacuolar sorting signal, we have prepared a chimeric gene containing the region encoding the barley lectin (BL) CTPP fused with the gene encoding the secreted protein, cucumber chitinase. Organelle fractionation and EM immunocytochemistry have demonstrated that the cucumber chitinase-CTPP fusion protein is redirected to the plant vacuole, confirming that the CTPP is a vacuolar sorting determinant. To analyse the functional elements of the CTPP which are necessary for proper sorting and processing, several mutants of the CTPP were prepared and have been analysed transiently and in transgenic tobacco plants. Preliminary data obtained from transient expression of these mutant constructs suggest that two independent short hydrophobic amino acid stretches of the BL CTPP are sufficient for correct sorting of BL to the vacuole. Neither charge nor glycosylation of the CTPP are necessary for targeting. In addition, our results have shown that the predicted amphipathic alpha-helix is not required for recognition of the sorting machinery.
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