Abstract
The biosynthesis and processing of wheat-germ agglutinin (WGA) were studied in developing wheat (Triticum aestivum L. cv. Marshall) embryos using pulse-chase labeling, subcellular fractionation and immunocytochemistry. A substantial amount of newly synthesized WGA was organelle-associated. Isolation of WGA on affinity columns of immobilized N-acetylglucosamine indicated that it was present in a dimeric form. When extracts from embryos pulse-labeled with [35S]cysteine were fractionated on an isopycnic sucrose gradient, radioactivity incorporated into WGA was detected at a position coincident with the endoplasmic reticulum (ER) marker enzyme NADH-cytochromec reductase. The WGA in the ER could be slowly chased into the soluble, vacuolar fraction, with a half-life of approx. 8 h. Immunolocalization studies demonstrated the accumulation and distribution of WGA throughout the vacuoles.
Four forms of the WGA monomer were characterized using immunoaffinity purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In-vitro translation of polyadenylated RNA isolated from developing wheat embryos produced a polypeptide with Mr 21 000. In-vivo labeling of embryos with radioactive amino acids resulted in the formation of a polypeptide of Mr 23 000 and the mature monomer of Mr 18000. When [3H]mannose was used in labeling studies, only the polypeptide of Mr 23 000 was detected. In-vivo labeling in the presence of tunicamycin yielded an additional polypeptide of Mr 20 000. These results indicate that WGA is cotranslationally processed by the removal of a signal peptide and the addition of a glycan, presumably at the carboxy-terminus (N.V. Raikhel and T.A. Wilkins, 1987, Proc. Natl. Acad. Sci. USA 84, 6745–6749). The glycosylated precursor of WGA is post-translationally processed to the mature form by the removal of a carboxyl-terminal glycopeptide.
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Abbreviations
- ER:
-
endoplasmic reticulum
- IgG:
-
immunoglobulin G
- Mr :
-
relative molecular mass
- poly(A)+RNA:
-
polyadenylated RNA
- SDS-PAGE:
-
sodium dodecyl sulfatepolyacrylamide gel electrophoresis
- WGA:
-
wheat-germ agglutinin
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Mansfield, M.A., Peumans, W.J. & Raikhel, N.V. Wheat-germ agglutinin is synthesized as a glycosylated precursor. Planta 173, 482–489 (1988). https://doi.org/10.1007/BF00958961
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DOI: https://doi.org/10.1007/BF00958961