Saporins - Type 1 Ribosome-inactivating proteins from soapwort (saponaria officinalis L.)

Abstract

Ribosome-inactivating proteins (RlPs) are found distributed throughout the plant kingdom. These proteins possess a RNA N-glycosidase activity whereby the depurination of a specific adenine residue from the large ribosomal subunit renders eukaryotic and, in some cases, prokaryotic ribosomes inactive. RIPs are most commonly found as single chain polypeptides and have been designated 'type I’. Some RIPs, however, are comprised of two polypeptide chains ('type 11'), an active polypeptide, analogous to the type I RIP, and a galactose-binding polypeptide. This thesis describes the characterisation of saporins, type I RIPs from soapwort (Saponaria officinalis L.). The work describes an analysis of saporin gene expression and the distribution of these proteins throughout the soapwort plant. Saporin gene expression in soapwort leaf tissue was shown to be temporally and spatially regulated. Saporins were distributed throughout the plant, the most abundant source of saporins was found in the seed tissue. The distribution of saporins in the seeds and leaves of soapwort was also studied at the cellular and subcellular level. Saporins were distributed to both vacuolar and extracellular sites of deposition in developing and mature seeds and to an extracellular site only in young leaves. Together with an analysis of the RIP activity of saporins on endogeneous ribosomes, where Saponaria ribosomes were shown to be depurinated by saporins, this work confirmed the assumption that saporins were targeted to a subcellular compartment separate from the cell cytosol where they could not come into contact with their own ribosomes. Molecular and biochemical differences between the leaf and seed saporin isoforms were also examined. The leaf saporins were shown consistently to be of a higher molecular weight than the seed saporins. This difference was also observed in other tissues of the soapwort plant. Differential extractability properties of the saporins were investigated and were applied to the purification of a leaf saporin isoform. The purified leaf saporin was identified as a new and uncharacterised isoform identical in its N-terminal sequence to a previously characterised seed saporin isoform.