Transport and posttranslational processing of the vacuolar enzyme α-mannosidase in jack-bean cotyledons.
α-Mannosidase (EC 3.2.1.24) is a vacuolar enzyme which occurs abundantly in the cotyledons of the jack-bean (Canavalia ensiformis (L.) DC). The mature enzyme is a tetramer with two polypeptides each of relative molecular mass (Mr) 66000 and Mr 44000. The enzyme has an interesting molecular structure because in its native form, it does not bind to concanavalin A (ConA) in spite of the presence of a high-mannose glycan. α-Mannosidase is synthesized in the developing cotyledons of jack-beans at the same time as the abundant proteins canavalin and ConA. The enzyme is synthesized as a precursor which has an Mr of 110000 and is associated with the endoplasmic reticulum (ER). Antibodies against the deglycosylated subunits cross-react with the Mr-110000 precursor. Processing of the precursor to the constituent polypeptides occurs posttranslationally, probably in the protein bodies. Immunocytochemical evidence shows that α-mannosidase is present in the ER and the Golgi complex of developing cells, and accumulates in the protein bodies.Labeling with [(3)H]glucosamine shows that after processing only the Mr-66000 polypeptide has glucosamine-containing glycans. The synthesis of these glycans is inhibited by tunicamycin, indicating that they are asparagine-linked oligosaccharides. Analysis of the glycans shows that there is a large glycan that is retained by ConA and a small glycan that is not retained by ConA. The large glycan is only partially sensitive to α-mannosidase because of the presence of a terminal glucose residue. Cross-reaction of the large subunit with an antiserum directed against small, complex glycans of plant glycoproteins indicates that this polypeptide probably has a xylose-containing glycan. Pulse-chase experiments carried out in the presence of tunicamycin show that the presence of glycans is not required for transport of α-mannosidase out of the ER-Golgi system.
Department of Biology, University of California San Diego, 92093-0016, La Jolla, CA, USA.
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Faye
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Greenwood
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Herman
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Sturm
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Chrispeels