Role of propeptide glycan in post-translational processing and transport of barley lectin to vacuoles in transgenic tobacco.
Mature barley lectin is a dimeric protein composed of two identical 18-kilodalton polypeptides. The subunits of barley lectin are initially synthesized as glycosylated proproteins, which are post-translationally processed to the mature protein preceding or concomitant with deposition of barley lectin in vacuoles. To investigate the functional role of the glycan in processing and intracellular transport of barley lectin to vacuoles, the sole N-linked glycosylation site residing within the COOH-terminal propeptide of barley lectin was altered by site-directed mutagenesis. cDNA clones encoding wild-type (wt) or glycosylation-minus (gly-) barley lectin preproproteins were placed under the transcriptional control of the cauliflower mosaic virus 35S promoter and introduced into Nicotiana tabacum cv Wisconsin 38. Barley lectin synthesized from both the wt and gly- constructs was processed and correctly targeted to vacuoles of tobacco leaves. Localization of barley lectin in vacuoles processed from the nonglycosylated gly- proprotein indicated that the high-mannose glycan of the barley lectin proprotein was not essential for targeting barley lectin to vacuoles. However, pulse-chase labeling experiments demonstrated that the glycosylated wt proprotein and the nonglycosylated gly- proprotein were differentially processed to the mature protein and transported from the Golgi complex at different rates. These results implicate an indirect functional role for the glycan in post-translational processing and transport of barley lectin to vacuoles.
Wilkins
T A
TA
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing 48824-1312.
Bednarek
S Y
SY
Raikhel
N V
NV
D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D001467:Hordeum / Q000235:genetics; D037102:Lectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D037121:Plant Lectins; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D010947:Plants, Toxic; D011134:Polysaccharides / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics; D014617:Vacuoles / Q000378:metabolism*
D037102:Lectins; D037121:Plant Lectins; D011134:Polysaccharides; D011498:Protein Precursors; D011994:Recombinant Proteins; C076978:barley lectin