Vacuolar targeting and posttranslational processing of the precursor to the sweet potato tuberous root storage protein in heterologous plant cells

Abstract

Sporamin, the tuberous root storage protein of the sweet potato, which is localized in vacuoles, is synthesized as a prepro-precursor with an N-terminal sequence of amino acids that includes a signal peptide and an additional pro-segment of 16 amino acids. A full-length cDNA for sporamin was placed downstream of the 35 S promoter of cauliflower mosaic virus and introduced into tobacco and sunflower genomes by Ti plasmid-mediated transformation. A polypeptide of nearly the same size as mature sporamin from the sweet potato was detected in transformed calli of tobacco and sunflower, as well as in the leaves, stems, and roots of regenerated, transgenic tobacco plants. Amino acid sequence analysis of the nearly mature-sized form of sporamin from the transformed tobacco cells revealed that it is actually longer by three amino acids at its N terminus than authentic sporamin purified from the sweet potato. By pulse labeling of suspension-cultured tobacco cells with [35S]methionine, the pro-form of the precursor to sporamin, but not the prepro-precursor, was detected. The 35S-labeled proform was chased to the nearly mature-sized form via an intermediate form which is slightly larger than the nearly mature-sized form. Analysis by Edman degradation of the intermediate form that was labeled in vivo with [3H]histidine suggested that it is longer by two amino acids at its N terminus than the nearly mature-sized form of sporamin. These results suggest that at least two steps of posttranslational processing of the pro-form occurs sequentially in tobacco cells. The posttranslational processing of the pro-form of the precursor to sporamin was inhibited by monensin, suggesting that this step takes place in the acidic compartment, probably in the vacuole. All of the sporamin polypeptides synthesized in transformed tobacco cells were retained inside the cell and sporamin was localized in the vacuole, as judged from results of subcellular fractionation. These results indicate that sporamin is appropriately targeted to the vacuole in tobacco cells.