Istituto Sperimentale per la Cerealicoltura

The b-32 protein is a functional ribosome inactivating protein

--M. Maddaloni, S. Lohmer1, I. Mauri2, E. Martegani2, F. Salamini1, R. Thompson1 and M. Motto

1Max-Planck-Institut für Züchtungsforschung, Köln

2Ist. Biochimica Comparata, Facoltà di Scienze, Univ. Milano

b-32 is one of the more abundant albumins accumulated specifically in the endosperm of maize developing kernels. The gene encoding this protein is under the control of the O2 and O6 loci. In a recent work we have reported that the product of the O2 locus is a strong transcriptional activator of the promoter of the b-32 gene (Lohmer et al., EMBO J. 10:617-624, 1991).

A search for homology to available protein sequences revealed that the b-32 peptide has a significant homology to the protein-synthesis inhibitor II from barley grains (Lohmer et al., quoted). This barley protein belongs to a group of proteins whose members, collectively called ribosome inactivating proteins (RIPs), act as severe inhibitors of eukaryotic protein synthesis by enzymatically cleaving the N-glycosyl bond of a specific adenine in the 28S rRNA, such that the elongation factor 2 binds inefficiently.

b-32 is shown to be a functional RIP by the criteria of inhibition of an in vitro rabbit reticulocyte cell-free translation system, and by specific N-glycosydase activity on 28S rRNA (Maddaloni, M et al., J. Genet. Breed., 1991, in press). To demonstrate in vivo the RIP activity of b-32 we have transformed the yeast Saccharomyces cerevisiae with a plasmid expressing the b-32 coding region under the control of an inducible promoter. The results showed that i) after induction the yeast cells arrest their growth, ii) protein synthesis is severely inhibited, and iii) the 28S rRNA is modified in the expected way. In addition, we have noted that in an in vitro transient gene expression assay with tobacco protoplasts as described in Lohmer et al. (quoted), the b-32 protein potentiates the expression of a reporter gene driven by a zein promoter.

Taken together, these experiments suggest that the b-32 protein might have a dual function. The first function appears to be related to the protection of the developing endosperm and/or the germinating seedlings against pathogen attacks. This role is also supported by the empirical observations that o2 and o6 mutant kernels are more vulnerable than wildtype kernels to ear-rotting diseases. The second function seems to be associated with the translation machinery of the maize endosperm in enhancing zein synthesis. In conclusion, the notion that b-32 shows a RIP activity on lower eukaryotes, opens up new possibilities in using this protein as a protectant agent against fungal infections in tissues and plants different than those in which its synthesis naturally occurs.

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