ENGINEERING IN-VIVO INSTABILITY OF FIREFLY LUCIFERASE AND ESCHERICHIA-COLI BETA-GLUCURONIDASE IN HIGHER-PLANTS USING RECOGNITION ELEMENTS FROM THE UBIQUITIN PATHWAY

The ubiquitin pathway targets proteins for degradation through the pos t-translational covalent attachment of the 76 amino acid protein ubiqu itin to epsilon-amino lysyl groups on substrate proteins. Two instabil ity determinants recognized by the ubiquitin pathway in Saccharomyces cerevisiae have been identified. One is described by the N-end rule an d requires specific destabilizing residues at the substrate protein N- termini along with a proximal lysyl residue for ubiquitin conjugation. The second is a linear uncleavable N-terminal ubiquitin moiety. The a bility of these two determinants to function in higher plants was inve stigated in tobacco protoplast transient transfection assays using DNA encoding variants of well characterized reporter enzymes as substrate s: firefly luciferase that is localized to peroxisomes (pxLUC), a cyto solic version of LUC (cLUC), and Escherichia coli beta-glucuronidase ( GUS). cLUC with phenylalanine encoded at its mature N-terminus was 10- fold less abundant than cLUC with methionine at its mature N-terminus. GUS with phenylalanine encoded at its mature N-terminus was 3-fold le ss abundant than GUS with methionine at its mature N-terminus. The pre sence of a uncleavable N-terminal ubiquitin fusion resulted in 50-fold lower protein accumulation of cLUC, but had no effect on GUS. Both in stability determinants had a much larger effect on cLUC than on pxLUC, suggesting that these degradation signals are either unrecognized or poorly recognized in the peroxisomes.