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 post-translational covalent attachment of the 76 amino acid protein ubiquitin to epsilon-amino lysyl groups on substrate proteins. Two instability determinants recognized by the ubiquitin pathway in Saccharomyces cerevisiae have been identified. One is described by the N-end rule and 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 ability of these two determinants to function in higher plants was investigated in tobacco protoplast transient transfection assays using DNA encoding variants of well characterized reporter enzymes as substrates: firefly luciferase that is localized to peroxisomes (pxLUC), a cytosolic 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 less abundant than GUS with methionine at its mature N-terminus. The presence of a uncleavable N-terminal ubiquitin fusion resulted in 50-fold lower protein accumulation of cLUC, but had no effect on GUS. Both instability 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.
Worley
C K
CK
Section of Molecular and Cellular Biology, University of California-Davis, 95616, USA.
Ling
R
R
Callis
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001517:Coleoptera / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008715:Methionine; D008830:Microbodies / Q000201:enzymology; D008969:Molecular Sequence Data; D010649:Phenylalanine; D010947:Plants, Toxic; D011523:Protoplasts; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae; D014026:Nicotiana / Q000201:enzymology*; D014162:Transfection; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D010649:Phenylalanine; D008715:Methionine; D008156:Luciferases; D005966:Glucuronidase; D008239:Lysine