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.
to the MNL 64 On-Line Index
Return to the Maize Newsletter Index
Return to the Maize Genome Database Page