Plant Physiol. (1988) 87, 420-426 0032-0889/88/87/0420/07/$01 .00/0 Glycoprotein Synthesis in Maize Endosperm Cells THE NUCLEOSIDE DIPHOSPHATE-SUGAR: DOLICHOL-PHOSPHATE GLYCOSYLTRANSFERASES Received for publication September 14, 1987 and in revised form January 4, 1988 WALTER E. RIEDELL' AND JAN A. MIERNYK* Seed Biosynthesis Research Unit, United States Department of Agriculture, Agricultural Research Service, Northern Regional Research Center, Peoria, Illinois 61604 ABSTRACT dolichol (24). Studies with mammalian cells and yeast (16, 32) have shown Microsomal membrane preparations from maize (Zea mays L., inbred that the enzymes of the dolichol cycle are associated with the A636) endosperm cultures contained enzymes that transferred sugar moie- ER. The assembly of Man,GlcNAc2-PP-dolichol is thought to ties from uridine diphosphate-N-acetylglucosamine, guanosine diphos- take place on the cytoplasmic surface of the ER. Subsequently, phate-mannose, and uridine diphosphate-glucose to dolichol-phosphate. this oligosaccharide is translocated to the lumen of the ER where These enzyme activities were characterized with respect to detergent and additional Man- and Glc-residues are transferred from lipid-car- pH optima, substrate kinetic constants, and product and antibiotic in- riers, forming the final tetradeccasaccharide-PP-lipid (21, 33). hibition constants. It was demonstrated by mild acid hydrolysis and high The oligosaccharide is then transferred en bloc from the lipid performance liquid chromatography that the products of the N-acetyl- carrier to the nascent polypeptide in a cotranslational event (21). glucosamine transferases were N-acetylglucosamine-pyrophosphoryl-dol- The first steps of oligosaccharide processing (e.g. removal of ichol and N,N'-diacetyl-chitobiosyl-pyrophosphoryl-dolichol and that the terminal glucose residues and, in mammalian cells, at least one product of the mannose transferase was mannosyl-phosphoryl-dolichol. mannose residue) also occur within the ER (16). Subsequent A large proportion of the products of the glucose transferase activity was steps of oligosaccharide processing and maturation take place in stable to mild acid hydrolysis. However, the proportion that was labile the Golgi apparatus and vacuoles was identified as glucosyl-phosphoryl-dolichol. Rate zonal sedimentation Maize endosperm cells cultured in vitro secrete acid hydrolases and isopycnic banding in linear sucrose density gradients in the presence into the culture medium (27). All of the hydrolases appear to of 1 millimolar ethylenediaminetetraacetic acid indicated that the glyco- be glycoproteins. These cells offer an excellent system with which syltransferase activities were located in the endoplasmic reticulum. The to examine protein glycosylation and its potential role in the glycosyltransferases were not solubilized by 500 millimolar KCI or by secretory process. As an initial step we have characterized the sequential washes with tris-(hydroxymethyl)aminomethane and water, NDP2-glycose: dolichol-P glycosyltransferases and determined treatments that release peripheral membrane proteins. Solubilization was their subcellular localization. achieved with low concentrations of Triton X-100. When sealed micro- somal vesicles were incubated with trypsin for 30 minutes in absence of detergent, the activity of N-acetylglucosaminyl-transferase was substan- MATERIALS AND METHODS tially reduced, while the activity of the glucosyl-transferase was somewhat Reagents. New England Nuclear3 provided the radioisotopes reduced. Activity of the mannosyl-transferase was resistant to inactivation UDP-[6-3H]GlcNAc (20.4 Ci/mmol), GDP-[1-3H]mannose (10.9 by incubation with trypsin unless Triton was present. Ci/mmol), and UDP-[1-3H]glucose (10.4 Ci/mmol). Dolichol- phosphate, unlabeled sugar nucleotides, BSA, trypsin, soybean trypsin-inhibitor, and tunicamycin were from the Sigma Chem- ical Company. Buffers were from Research Organics, Inc. Se- pharose-4B was from Pharmacia. Genzyme Corporation supplied the (+ )-1-deoxynojirimycin. Ecoscint scintillation fluid was from The oligosaccharides of N-linked glycoproteins are assembled Research Diagnostics. BioRad supplied the reagents for protein from lipid-linked sugar intermediates by the membrane-bound quantitation. All materials were of the highest grade commer- enzymes of the dolichol pathway (8). Formation of the lipid- cially available. linked intermediates is achieved by the transfer of GlcNAc, Man, Tissue Culture. The suspension cultures were originally de- and Glc from nucleotide donors to lipid carriers (31). In the rived from 6- to 10-d-old maize (Zea mays L., inbred A636) initial step of the pathway, GlcNAc-1-P is transferred from UDP- endosperm. The medium, culture conditions, and transfer regime GlcNAc to dolichol-P, forming GlcNAc-PP-dolichol. A second have been previously described (27). GlcNAc is then donated by UDP-GlcNAc, forming N,N'-diace- Enzyme Assays. Glucan synthetase I and IDPase were assayed tylchitobiosyl-PP-dolichol (16). Mannose residues which make by the method of Green (13). To determine the latent increment up the heptasaccharide core are transferred directly from GDP- in IDPase activity, the initial enzyme activity was subtracted from Man to this disaccharide-lipid (4). The four outer Man residues the total activity after 3 d of storage at 4°C (28). Other marker are donated by Man-P-dolichol, synthesized from GDP-Man and enzymes were assayed by established procedures (26). dolichol-P. Finally, three terminal Glc-residues are transferred Glycosyltransferase activites were assayed by measuring the from Glc-P-dolichol, synthesized from UDP-Glc and dolichol-P, conversion of radioactive sugars from nucleoside diphosphate- forming the lipid-linked oligosaccharide Glc3Man,GlcNAc2-PP- 2 Abbreviation: NDP, nucleoside-diphosphate. I Present address: United States Department of Agriculture, Agri- 3Names of vendors are included for the benefit of the reader and do cultural Research Service, Northern Grain Insects Research Laboratory, not imply endorsement or preferential treatment by the United States RR No. 3, Brookings, SD 57006. Department of Agriculture. 420 MAIZE ENDOSPERM GLYCOSYLTRANSFERASES 421 sugars into products soluble in chloroform:methanol 2:1, v/v). concentration of 0.01%. After brief mixing, trypsin was added Solvent was removed from 20 ,g of dolichol-P by evaporation to each tube to a final concentration of 30 Ag mg protein- '. The with a stream of N2-gas. A solution (450 Al final volume) con- tubes were incubated on ice and aliquots were removed at the taining 0.015% Triton X-100, 50 mM TES (pH 7.0), 10 mM MgCl2 indicated times. A 10-fold excess of soybean trypsin inhibitor plus 50 or 100 Al of membrane preparation was added. Reaction was immediately added to each aliquot prior to assay of enzyme was initiated by addition of 50 ,u of 60 ,mM NDP-sugar (200,000 activity. In parallel with the isolation of microsomal vesicles for dpm). After mixing, the assays were conducted at 30°C. Under enzyme assays, a tissue sample was homogenized in the presence these assay conditions, the reactions were linear for at least 20 of 10 ,uCi of 3H-inulin. Some of the inulin was entrapped during min. Reactions were stopped by the addition of 2.5 ml chloro- the formation of vesicles by fragmentation of the endomembrane form:methanol (2:1) and, after mixing, the lower organic phase network. Release of radioactivity from the vesicles can serve as was removed. The aqueous phase was reextracted, the combined a sensitive measure of membrane integrity during in vitro pro- organic phases were pooled and washed with chloroform: teolysis. After treatment, the vesicles were sedimented in the methanol:H2O (3:48:47, v/v/v), then the lower organic phase was ultracentrifuge, and the proportion of radioactivity in the su- removed for liquid scintillation spectrometry. When inhibitors pernatant and pellet was quantitated by liquid scintillation spec- of the enzyme activities were tested, they were preincubated in trometry. Treatment of the vesicles with Triton X-100 released the assay mixture for 1 min prior to addition of the NDP-sugar. all of the entrapped inulin. Using this method, it was found that It was necessary to add 2 mm DTT and 20 /LM deoxynojirimycin stability of the isolated maize vesicles during in vitro manipu- to assay mixtures in order to reliably assay Glc-transferase ac- lation was always greater than 90%. tivity. Enzyme Solubilization. Microsomal membrane fractions were Product Analysis. Mild acid hydrolysis of the products of the resuspended in 100 mm TES (pH 7.5). One-ml aliquots (1.5 mg glycosyl transferase reactions was performed after pooling the membrane protein) were placed into centrifuge tubes, and Triton washed chloroform:methanol 2:1 fractions from four separate X-100 or KCl was added to the indicated final concentrations. reaction mixtures. One ml was removed for scintillation count- The tubes were gently shaken on ice for 20 min. Membranes ing. The remaining extract was dried under a stream of N2, were subsequently pelleted by centrifugation at 200,000g using dissolved in 1 ml 10 mm HCl in 50% propanol, and then incu- a TLA-100.2 rotor in a Beckman TL-100 ultracentrifuge. Mem- bated at 100°C for 20 min. After cooling, the hydrolysate was brane pellets were resuspended in buffer prior to assaying for mixed with 1 ml chloroform, and the phases were separated enzyme activity. before removal of aliquots for scintillation counting. The aqueous Other Analytical Methods. Sucrose