Expression of engineered zein polypeptides in yeast --B. Basso, L. Bernard, P. Ciceri and A. Viotti It is commonly accepted that zein proteins consist of four domains: a head region of 30-40 amino acids, a central part of 8-9 repeats of about 15 amino acids each, separated by 2 or more glutamine residues, and a short tail of 8-10 amino acids.

In order to test the biological and functional importance of the various domains for the structure of the protein and for its aggregation into protein bodies, the yeast S. cerevisiae was employed as a heterologous system for the expression of the whole zein sequence coding for a 24 Kd protein (M6, Viotti et al., EMBO J. 4, 1985) or of truncated sequences representing only specific parts of this gene. The maize sequences were inserted in an E. coli/yeast shuttle vector (pEMBLyex2), under the yeast CYC1 gene promoter and under the control of the UAS-GAL enhancer. When a truncated gene had to be expressed, a modified form of pEMBLyex2 was employed, carrying an "ATG" starting codon in the vector insertion site.

The constructions to be tested (pyM6 = the whole gene; pyM6 T/H = the gene lacking the sequence coding for the signal peptide; pyM6P/H = the gene lacking the sequences coding for the signal peptide, the head region and the two first repeats) were used for yeast transformation. Yeast cells were grown in galactose medium and the cell content was then analyzed by SDS-PAGE following specific zein extraction; in each case the presence of zein was clearly detectable. Cells transformed with pyM6 and pyM6P/H were also analyzed by immuno-cytological detection experiments. Confirming previous results (I. Coraggio et al., EMBO J. 5, 1986 and Eur. J. Cell Biol. 47, 1988), in cells expressing the whole zein sequence, the protein was found localized mainly at the E.R. level, while, where a truncated sequence was expressed, the protein was localized in mitochondria, aggregated in protein body-like structures.

The microsequence analyses of the amino terminal regions of all the zein proteins extracted from the recombinant yeasts containing the entire genes reveal a regular processing in the maturation of the various signal peptides. Similar analyses of the polypeptides from the truncated genes showed that they were unprocessed. Other constructions, such as further shortened sequences or different lysine-inserted zeins, are under examination to complete and define the evidence here reported.

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