Subcellular Distribution of Membrane‐Bound Glycosyltransferases from Pea Stems

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Abstract
Particulate and subcellular membrane preparations from growing regions of etiolated pea stems catalyse the transfer of sugars from UDP‐[14C]glucose and GDP‐[14C]mannose to a variety of endogenous lipid, glycoprotein and polysaccharide acceptors. Glycolipids were fractionated and identified by chromatography on DEAE‐cellulose and silica gel. They included neutral components, e.g. sterylglycosides, and polar lipids, comprising polyprenylmonophospho‐monosaccharides and polyprenyldiphospho‐oligosaccharides. High‐molecular‐weight material was partially hydrolyzed with pronase or acidic cyanogen bromide in order to solubilize glycoproteins and products bound to proteins, and to separate these from insoluble polysaccharide.Pea membranes with densities at which endoplasmic reticulum equilibrates in linear sucrose gradients contained most of the recovered capacity for glycosylation of endogenous polyprenyl monophosphate. When dolichyl phosphate was added to the membrane preparations, it was readily glycosylated in the presence of lysophosphatidylcholine but there was no indication that dolichylphospho‐monosaccharide served as an intermediate for synthesis of other products. In contrast, glycosyltransfer to endogenous neutral lipids, polyprenyl diphosphate and polymeric products occurred to a limited extent in the endoplasmic reticulum region and was most extensive in membranes which equilibrate at higher densities, i.e. in regions containing Golgi and possibly plasma membrane vesicles. The data are consistent with the conclusion that glycosylation of polymeric products occurs throughout the pea endomembrane system, with polyprenylmonophospho‐monosaccharide available to act as an intermediate (precursor) primarily in the endoplasmic reticulum.