Subcellular Localization of Glycosidases and Glycosyltransferases Involved in the Processing of N-Linked Oligosaccharides

Abstract

Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc3Man,(GlcNAc)2, was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associated with the Golgi. These include mannosidase I (removes 1-2 mannose residues from Man6 4GlcNAcl2), mannosidase II (removes mannose residues from GlcNAcMans5GlcNAcI2), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). We have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltransferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities.Recent evidence from several laboratories indicates that plant glycoproteins contain both high mannose and complex glycans attached to the polypeptide via a GlcNAc -. asparagine bond. The high mannose glycans have the general structure Man5-9 (GlcNAc)2; the complex glycans usually have fewer mannose residues, additional GlcNAc, and other sugars, such as fucose, xylose, and/or galactose (4). Glc3Man9(GlcNAc)2 to Man9(GlcNAc)2; a-mannosidase I, which removes up to four a 1-2 linked mannose residues from Mang(GlcNAc)2 to yield Man5-8(GlcNAc)2; and a-mannosidase II, which removes an a 1-3 and a 1-6 mannose from GlcNAc Man5(GlcNAc)2 to produce GlcNAcMan3(GlcNAc)2. The various glycosyltransferases transfer sugars (GlcNAc, fucose, xylose, and galactose) from their nucleoside diphosphate donors to specific N-linked glycan acceptors in a preferred sequence. GlcNAc-transferase I precedes while GlcNAc transferase II follows the action of a-mannosidase II to generate GlcNAcMan5(GlcNAc)2 and (GlcNAc)2Man3(GlcNAc)2, respectively. The latter product is an excellent acceptor for both fucosyl and xylosyltransferase (15), and presumably also for galactosyltransferase. Figure 1 shows the structure of the complex oligosaccharide that would result from the glycan-processing steps described above.The subcellular locations of several of these glycan-processing enzymes in plants have been ascertained indirectly through characterization of the oligosaccharides obtained from, and ...

Complementation of an Arabidopsis thaliana mutant thatlacks complex asparagine-linked glycans with the human cDNA encodingN-acetylglucosaminyltransferase I.

“…ulated cisternae) are found in sucrose gradients, and other known Golgi enzymes also band at this density (13 (21). The finding that this human membrane protein is confined to the Golgi apparatus of plant cells is in contrast to results obtained with the G protein of vesicular stomatitis virus.…”

Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum

“…Both antisera were obtained by injecting rabbits with a purified secreted plant protein. Proteins having this carbohydrate group have been processed through the Golgi apparatus (18). Figure 3 shows that phosphate starvation induced an increase in secretion of some Golgi-processed media proteins.…”

“…The paper was cut and immunoblotted with either AP3 or a polyclonal antiserum designated 949 (generously provided by M. J. Chrispeels, UC San Diego) that reacted specifically with N-linked oligosaccharides that contain xylose (8). This indicated processing through the Golgi-apparatus ( 18). It may be seen that the staining patterns for the two antisera were almost identical.…”

Functional Compartmentation of the Golgi Apparatus of Plant Cells

“…The fact that all plant Golgi cistemae possess very similar densities, together with the lack of proven enzymic markers, has hampered efforts to study subcompartmentation of Golgi stacks derived from plant cells (4,42), even though they exhibit a pronounced morphological polarity in electron microscopic images (36). Furthermore, because the structure of many types of complex polysaccharides is still poorly defined, it is difficult to even contemplate in a more than superficial manner how the glycoprotein and complex polysaccharide synthesis pathways might be integrated in plants.…”