In vitro mutated phytohemagglutinin genes expressed in tobacco seeds: role of glycans in protein targeting and stability.
Phytohemagglutinin is a glycoprotein that accumulates in the protein storage vacuoles of bean seeds. The mature glycoprotein has a high-mannose and a complex glycan. We describe here the use of site-directed mutagenesis and expression of the mutated genes in transgenic tobacco to study the role of glycans in intracellular targeting. The reading frame for phytohemagglutinin-L was mutated so that either one or both of the glycosylation signals were disrupted to specifically prevent the attachment of asparagine-linked glycans. Expression of these genes with the beta-phaseolin promoter in the seeds of transgenic tobacco plants showed that phytohemagglutinin-L with only one glycan or without glycans was correctly targeted to the protein storage vacuoles of the seeds. Furthermore, the absence of either the complex glycan or the high-mannose glycan did not alter the processing of the other glycan. On the basis of these results, we propose that the targeting signal of this vacuolar protein is contained in its polypeptide domain and not in its glycans.
Voelker
T A
TA
Department of Biology, University of California, San Diego, La Jolla 92093-0116.
Herman
E M
EM
Chrispeels
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D004247:DNA; D006031:Glycosylation; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010835:Phytohemagglutinins / Q000235:genetics* / Q000378:metabolism; D037121:Plant Lectins; D010940:Plant Proteins / Q000235:genetics; D010947:Plants, Toxic; D011134:Polysaccharides / Q000502:physiology*; D011401:Promoter Regions, Genetic; D014026:Nicotiana / Q000196:embryology / Q000235:genetics* / Q000648:ultrastructure; D014617:Vacuoles / Q000378:metabolism
D010835:Phytohemagglutinins; D037121:Plant Lectins; D010940:Plant Proteins; D011134:Polysaccharides; C048826:phaseolin protein, Phaseolus vulgaris; D004247:DNA