We propose to map the protein determinants which are likely to be responsible for target specification and sorting of the seed storage protein phaseolin. This will be approached by producing deletions in the coding sequence of a cloned phaseolin gene and by constructing phaseolin/GUS gene chimeras, which will be subsequently transferred into tobacco by using Ti plasmid- mediated transformation techniques. Analysis of the expression and localization of the different constructs in transgenic seeds will then be performed, to establish a correlation between specific peptide sequences and targeting properties. N-linked glycosylation and sorting of phaseolin, will also be studied in an insect cell line infected with a baculovirus expression vector. We intend to eventually be able to relate the function of specific sequences on the polypeptide portion of the molecule and of oligosaccharides, to their structure. In studies contemplated in a separate proposal, the tertiary structure of the phaseolin protomers will be determined by X-ray crystallography.
