Subcellular Localization Glycosyl Transferases Involved in

Plant Physiol. (1968) 61, 451-459

Subcellular Localization of Glycosyl Transferases Involved inGlycoprotein Biosynthesis in the Cotyledons of Pisum sativum L.'

Received for publication June 22, 1977 and in revised form September 12, 1977

JERRY NAGAHASHI AND LEONARD BEEVERSDepartment of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019

ABSTRACT

Subcellular membrane fractions from 21-day-old pea (Pisum satdvum)cotyledons that have associated UDP-N-acetylglucosamine N-acetylglu-cosaminyl transferase and GDP-mannose mannosyl transferase activitieshave been isolated and identified. The rough endoplasmic reticulum (RER)is the principal location of glycosyl transferases involved in the assemblyof Upid-linked sugar intermediates and glycoproteins. Antimycin A-insen-sitive NADH-cytochrome c reductase activity was used to identify RERat a density of 1.165 g/cc in sucrose gradients. The high proportion ofRER in this fraction was confirmed by electron microscopy.

Other mannosyl transferases are found at a density of 1.123 g/cc and1.201 g/cc but these glycosyl transferases do not appear to be involvedwith the formation of lipid-linked sugar intermediates utilized in glycopro-tein biosynthesis.

Recent investigations have indicated that lipid-linked sugarintermediates are involved in the synthesis of some glycoproteinsin plants (2, 7, 8, 14), fungi (15), and animals (for review see 33).The formation of lipid-linked sugar derivatives and glycoproteinsis catalyzed by particulate fractions and various schemes havebeen developed to model the glycosylation process (16, 21). Sub-cellular localization of the required glycosyl transferases is essen-tial to support any model.Although information has been assembled concerning the sub-

cellular location of the glycosyl transferases involved in formationof cell walls in plants (10, 32) little data are available on the siteofglycoprotein glycosyl transferases. One report (4) has implicatedthe plasma membrane as the site of synthesis of lipid-linkedglucose intermediates involved in glycoprotein formation but thelocation of the transferase activity was uncertain. Membranefractions derived from the Golgi apparatus of carrot root (9) andonion stem (24) have been reported to contain glycoprotein gly-cosyl transferase activity. No glycosyl transferase was reported inthe ER and the importance of the Golgi apparatus in the glyco-sylation of glycoproteins was stressed (9, 24).No detailed analyses have been performed to determine the

site of glycosyl transferases required for synthesis of lipid-linkedsugar intermediates involved in initial synthesis of the glycopep-tide bond in animal systems (33) and plants. Since we haveestablished the role of lipid-linked intermediates in glycoproteinsynthesis in a particulate fraction from cotyledons ofPisum sativum(2), we have attempted to determine which membrane componentsin the particulate fraction catalyze glycosyl transfers to form lipid-linked sugars and glycoproteins. The approach used to localizethe transferases was a combination of differential centrifugation

' Research was supported by National Science Foundation GrantsBMS575-05722 PGM76-05722 AOl and by funds provided by the Univer-sity of Oklahoma Faculty Research Council.

and linear sucrose density gradient analysis in conjunction withelectron microscopy.

MATERIALS AND METHODS

Plant Material. Pea (Pisum sativum cv. Burpeana) seeds weregerminated and grown as described previously (1). Pea cotyledons21 days postanthesis were used for all experiments.

Cell Disruption and Differential Centrifugation. Seeds wereremoved from pods, testa and embryos were excised, and cotyle-dons were weighed (10-20 g fresh wt). All further manipulationswere at 0 to 4 C. Tissue was razor blade-chopped with an electri-cally powered, mechanically driven chopper (2) for 1 to 2 min ina grinding mix consisting of 0.5 M sucrose, 5 mM 2-mercaptoeth-anol, and 30 mM Tris-MES (pH 7.5). One ml of GM2 was used/gfresh wt but immediately after chopping, enough GM was addedto make the ratio 4:1. The homogenate was filtered throughcheesecloth and centrifuged at 250g for 5 min to remove cell walldebris, intact cells, starch, and intact chloroplasts and nuclei. Forthe data in Table I, the supernatant was successively centrifugedat l,000g for 10 min, 8,000g for 15 min, 13,000g for 15 min, 40,000gfor 35 min, and 80,000g for 35 min. Centrifugations up to 13,000gwere performed in a Sorvall SS-34 rotor and high speed centrifu-gations were performned in a Beckman 42.1 rotor. Pellets fromthese centrifugations were suspended in GM and pelleted at theirinitial force. Washed pellets were suspended in 0.25 M sucrose, 1mM 2-mercaptoethanol, and 1 mM Tris-MES (pH 7.2) (bufferedsucrose) and assayed for various enzyme activities. Proteins wereestimated by the procedure of Lowry et al. (19).

For linear sucrose gradient analysis, fractions pelleted between250 and 13,000g (crude mitochondrial pellet) and between 13,000and 40,000g (crude microsomal pellet) were split into equal halves.One half was washed in GM plus 5 mm EDTA and the otherhalf in GM minus EDTA and pelleted at their initial force. Pelletswashed in GM were "normal" treated controls while pelletswashed in GM plus 5 mm EDTA were called "EDTA"-treated.After washing, pellets were suspended in 2 ml of buffered sucroseand overlaid on identical 36-ml sucrose gradients.

Linear Sucrose Gradients. Density gradients of 15 to 60%7o(w/w) sucrose in I mM Tris-MES (pH 7.2) plus I mm 2-mercap-toethanol were made with an ISCO model 570 gradient maker(Instrumentation Specialties Co., Lincoln, Nebr.). Membrane pel-lets were layered over linear gradients and centrifuged to equilib-rium at 82,500g for 15 hr in a Beckman SW 27 rotor, fractionatedwith an ISCO model 182 density gradient fractionator into 30fractions (1.2 ml/fraction), and monitored with an ISCO model222 UV analyzer. Per cent sucrose was determined with an Abbe3L refractometer (Bausch and Lomb). Fractions were assayed forvarious enzyme activities and/or combined and prepared forelectron microscopy.

2 Abbreviations: GlcNAc: N-acetylglucosamine; GM: grinding mix;Man: mannose.

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NAGAHASHI AND BEEVERS

Table I. Distribution of various subcellular markers in membrane fractions obtainedby differential centrifugation of a pea cotyledon homogenate.

Cyt c Oxidase NADH-Cyt c Reductase CPM from CPM from1 2 - Antimycin A + Antimycin A GDPF-TC-Mannose UDP - '4C GlcNAc

SA TA SA TA SA TA INHIBITION SA TA SA TA.svl % amol% smol 7 % cpm/mg % cpm/mg %

FRACTION mg.min mg.min mRg.min

250-8000g 0.067 81.2 0.044 29.3 0.024 19.2 45.5 1955 29.2 193 14.08000-13,OOOg 0.051 11.9 0.143 18.1 0.116 17.5 18.9 6616 19.0 1465 20.4

13,000-40,OOOg 0.019 6.5 0.237 43.9 0.234 52.0 1.3 10,693 44.9 2655 54.140,000-80,0OOg 0.004 0.4 0.162 8.7 0.175 11.3 0.0 5679 6.9 1962 11.5

pH 6.5 Mg-ATPase +pH 7.5 IDPase Chlorophyll Protein

-K +Xr K -S timu la t ionSA TA SA TA SA TA SA TA SA lotal Total4Imol Pi % PmolPi % amol Pi % aImol Pi % tij m

FRACTION mg.hr mg.hr mg.hr mg.hr mg fraction %

250-80OOg 2.28 56.2 2.62 54.1 0.34 43.7 1.82 52.8 12.3 65.9 18.24 64.38000-13,000g 3.97 18.8 4.79 19.0 0.82 20.3 2.83 15.7 24.3 24.5 3.50 12.3

13,000-40,OOOg 2.79 19.4 3.72 21.7 0.93 33.5 2.95 24.0 5.9 8.5 5.13 18.240,000-80,OOOg 2.83 5.6 3.08 5.2 0.25 2.5 3.19 7.5 2.7 1.1 1.48 5.2

1SA; specific activity

2TA; total activity

Enzyme Assays. Adenosine triphosphatase (ATPase, pH 6.5and 9) and inosine diphosphatase (IDPase, pH 7.5) were assayedaccording to the method described by Leonard and Van DerWoude (17). Inorganic phosphate released was determined asdescribed by Hodges and Leonard (1 1).NADH-Cyt c reductase and Cyt c oxidase activities were assayed

spectrophotometrically by following the reduction or oxidationofCyt c at 550nm (1 1). NADH-Cyt c reductase activity was assayed± I ,LM antimycin A as described by Lord et al. (18) with NADHused to initiate the reaction.

Glycosyl transferase activities were determined by the transferof radioactivity from labeled nucleoside sugar phosphates intotrichloroacetic acid-precipitable material. Uridine diphospho-N-acetyl-D-[U'4CJglucosamine (300 mCi/mmol, 24,uCi/ml) and gua-nosine diphospho-[U"4C]Man (179 mCi/mmol, 25 ,uCi/ml) pur-chased from Amersham Searle Corporation were used as sub-strates to measure activities of N-acetylglucosaminyl transferasesand mannosyl transferases, respectively. Membrane fractions (0.3ml) were incubated with 0.1 ml of substrate mix (final concentra-tions of 50 mM Tris, 10 mM KCI, 10 mM MgCl2, and 2.5 mM 2-mercaptoethanol brought to pH 7 with HCI plus 1 pl of GDP-['4CJMan or I yl of UDP-[I4CIGlcNAc) in a shaking water bathat 38 C for I hr. One ml of a 2 mg/ml solution of BSA fractionV (Sigma Chemical Co., St. Louis, Mo.) was added to eachreaction tube when the reaction was stopped with trichloroaceticacid (5% w/v, final concentration). Precipitated material wasfiltered through glass fiber filters (Whatman GF/A) and rinsedthoroughly with cold 5% trichloroacetic acid to remove unincor-porated label.For detailed characterization of the glycosyl transferase reac-

tions presented in Table II, fractions collected from linear sucrosegradients between 1.11 and 1.13 g/cc, 1.16 and 1.17 g/cc, and1.19 and 1.21 g/cc were combined and incubated with labeledsubstrate. Reactions were stopped by addition of 0.7 ml of H20containing 2 mg ofBSA and 2.5 ml of chloroform-methanol (2:1).Further extraction of the chloroform-methanol-water (1:1:0.3)-soluble material and preparation of the residue were carried outas described previously (2). Residues were treated either withSDS or protease (2) and counted or radioactivity was determinedon untreated residues.Chl was extracted from membrane fractions with 80o acetone

and A at 652 nm was converted to jig of Chl (13).Electron Microscopy. Membrane fractions isolated from sucrose

gradients were centrifuged at 81,800g for 30 min (Beckman SW27.1 rotor) in 2.5% glutaraldehyde buffered in 0.1 M K-phosphatebuffer (pH 7.2). Cellulose-nitrate tubes containing the pellets wereplaced in ice and membrane fractions were fixed for an additionalhr. Pellets were rinsed with cold buffer for 45 min (several changesof buffer were made) and postfixed with buffered 1% Os04 for

Table II. Subcellular membrane fractions from pea cotyledons were collectedfrom a sucrose gradient centrifuged with a 13,000-40,000g pellet.Aliquots from isolated fractions were incubated with UDP-1 C GlcNAcor GDP-14C Mannose.

CPM from GDP- C Mannose

Counts retainedDensity Trichloroacetic Lipid extraction Lipid Free on filter paper

acid precipitate 2:1 1:1:0.3 Residue after SDS treatment

g/cc

1.123 2848 310 95 481 921.165 1352 579 143 208 381.201 1682 53 37 690 91

CPM from UDP-14C GlcNAc

1.123 547 307 89 112 01.165 1173 910 174 125 01.201 206 150 31 48 0

1.5 hr. Samples were dehydrated through a graded acetone seriesand embedded in Spurr's epoxy resin (31). Intact cotyledons weresliced into pie-shaped wedges, fixed at room temperature, andprepared for microscopy in the same fashion. Thin sections werestained with uranyl acetate-lead citrate (27) and viewed with aHitachi 1 lB-I electron microscope.

RESULTS

DIFFERENTIAL CENTRIFUGATION

Previous studies indicated that mannosyl transferases andGlcNAC transferases were associated with particulate fractionsfrom pea cotyledons (2). In the present study, a series of differ-ential centrifugations were performed to determine the centrifugalforce necessary to pellet most of the glycosyl transferase activities.The distribution of specific enzyme "markers" for plant organellesand membranes was also determined on the pellets.

Cyt c oxidase was used to locate mitochondria and Table Iindicates that 93% of the total enzyme activity of the crudehomogenate is sedimented between 250 and 13,000g. The highestspecific activity of Cyt c oxidase was also found in this fractionand agrees with the distribution of mitochondria reported previ-ously (12, 13, 17). Most ofthe Chl and protein sedimented between250 and 13,000 g (Table I). The high per cent of protein can beattributed to the sedimentation of protein bodies with their asso-ciated reserve proteins.

Latent IDPase activity has been used as a marker for Golgiapparatus membranes (26). Maximum nucleoside diP activity isachieved after storage at 0 to 2 C for 3 to 6 days (17, 26) withGolgi apparatus membranes usually isolated in postmitochondrialfractions. In the present investigation, over 60o of the IDPaseactivity sedimented between 250 and 13,000g (Table I); moreoverthe enzyme showed no latency. Nucleoside diP activity associated

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MEMBRANE-BOUND GLYCOSYL TRANSFERASES

with plasma membrane (13), other subcellular membranes ororganelles, or nonspecific phosphatases may account for the largepercentage of IDPase detected in the 250 to 13,000g fraction.Over 40%o of the K+-stimulated ATPase was sedimented by

centrifugation between 250 and 8,000g (Table I). This observationis inconsistent with previous reports (12, 13, 17) which demon-strated that the majority of the K+-stimulated ATPase associatedwith plasma membrane vesicles was sedimented by centrifugationabove 13,000g. Part of this apparent discrepancy may be attributedto the homogenization technique. In the present investigation,razor blade chopping was used and this produces less shearingforces than the mortar and pestle or Polytron homogenization(13). Larger vesicles of the plasma membrane produced by razorblade chopping would pellet under lower centrifugal forces incomparison to the higher forces necessary to sediment the smallervesicles produced during harsher isolation procedures (13). Therecovery of a large percentage of the K+-stimulated ATPase inthe 250 to 13,000g fraction is consistent with this concept.NADH-Cyt c reductase activity has been reported in the micro-

somal fraction of plants and is often used to identify the ER inplants (6, 17, 18, 22) and animals (5). NADH-Cyt c reductaseactivity has also been associated with microbodies (6), nuclearmembranes (22), and mitochondria (18). The mitochondrialNADH-Cyt c reductase, in contrast to that associated with theother membranes and organelles, is inhibited by antimycin A(18).Over 40o of the total NADH-Cyt c reductase activity in the

crude homogenate was pelleted by centrifugation between 13,000and 40,000g (Table I). This fraction had the highest specific activityand was not inhibited by antimycin A. The mitochondrial richmaterial sedimenting between 250 and 13,000g (Table I) alsocontained NADH-Cyt c reductase activity but this enzyme wasinhibited by antimycin A.The majority of the UDP-GlcNAc transferase activity was

pelleted between 13,000 and 40,000g (Table I). GDP-Man transfer-ase was also associated with this pellet, however considerableactivity also sedimented between 250 and 13,000g (Table I). Theco-sedimentation of UDP-GlcNAc transferase and mannosyltransferase activities with antimycin A-insensitive NADH-Cyt creductase between 13,000 and 40,000g suggested that the glycosyltransferases may be associated with the ER. Because NADH-Cytc reductase is not specifically associated with the ER, sucrosedensity centrifugation was performed to confirm the localizationof the glycosyl transferases.

LINEAR SUCROSE DENSITY GRADIENT ANALYSES OF THE 13,000TO 40,O00g PELLET

When a 13,000 to 40,000g pellet was centrifuged in a linearsucrose density gradient and assayed for UDP-GlcNAc transfer-ase, one major peak of activity was found between 1.15 and 1.18g/cc (Fig. 1). This is the density range reported for RER in plants(18, 25) and is coincident with the major peak of NADH-Cyt creductase activity (Fig. 1). When GDP-Man transferase was as-sayed, two distinct peaks of activity were found. The first peakat a density of 1.123 g/cc (Fig. 1) is coincident with the majorpeak of IDPase activity but a second distinct peak of activity at1.201 g/cc is of unknown origin. A third broad area of mannosyltransferase activity is found throughout the center of the gradientin the same density range as NADH-Cyt c reductase and UDP-GlcNAc transferase (Fig. 1). To confirm the association ofNADH-Cyt c reductase activity with ER and its coincidence withglycosyl transferase activities, further gradient analyses were per-formed.A survey ofantimycin A-sensitive NADH-Cyt c reductase activ-

ity in a gradient overlaid with a normal treated pellet indicatedthat the majority of the activity was insensitive to the antibiotic(Fig. 2C). However, a small inhibition of NADH-Cyt c reductase

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transferase and UDP-GlcNAc transferase isolated from pea cotyledonsand sedimented between 13,000 and 40,000g. A 2-ml sample ofresuspendedpellet was layered over a 36-ml sucrose gradient of 15 to 60% (w/w)sucrose. Centrifugation was for 15 hr at 82,500, (ray 11.83 cm). Fractionsize was 1.2 ml.

activity was consistently observed at 1.185 g/cc. This activity inthe absence of antimycin A occasionally appears as a separatepeak (Fig. 2C) but is most frequently indicated as a shoulder(Figs. 3 and 4).

If the microsomal pellet is washed with EDTA prior to sucrosedensity gradient centrifugation, the major area of protein is shiftedfrom a density of 1.16 g/cc to 1.10 g/cc (Fig. 2, C and D). Thereis a concomitant shift of the antimycin A-insensitive NADH-Cytc reductase, however a small amount of antimycin A-sensitiveactivity at 1.185 g/cc does not shift, indicating the presence ofcontaminating mitochondria. In contrast to the RER-associatedNADH-Cyt c reductase activity, mitochondrial associated activitydoes not change in density with EDTA treatment and is inhibitedby antimycin A (see next section).The change in density of the protein (Fig. 2, C and D) and

shift of antimycin A-insensitive NADH-Cyt c reductase activityindicate that RER (1.165 g/cc, Fig. 2C) is being converted to amembrane of lighter density or "smooth" ER (1.102 g/cc, Fig.2D) by EDTA treatment. This transition has been reported pre-viously for plant tissue (18, 25) and an EDTA concentration-dependent dissociation of ribosomal subunits from RER has beenreported in animal systems (28). EDTA apparently chelates Mg2+ions since addition of an appropriate concentration of Mg2+ toan EDTA-sucrose gradient will maintain ribosomal attachmentto ER (18). Our observation that the A trace (280 nm) shows lessprotein in the gradient following E-DTA treatment (compare Atraces for Fig. 2C and 2D) is consistent with the above reports.Increased protein is recovered in the supernatant of the EDTAwash compared to the normal wash (data not shown) and ispresumably derived from ribosomal components dissociated fromER.

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1.lOg/cc 1.185g/cc 1.10g/cc 1.16g/cc 1.185g/cc K+-stimulated Mg2+-ATPase activity has been associated withplasma membranes isolated from nongreen (12, 17) and greenplant tissue (13) and was used as a marker. In pea cotyledons,very low levels of ATPase activity were observed (Table I andFig. 5). The K+-stimulated ATPase showed four areas of activitywith the major peak at a density of 1.173 g/cc (Fig. 5A). AfterEDTA treatment of a microsomal pellet, the distribution of K+-stimulated ATPase activity in the sucrose gradient did not change(Fig. 5B). Since the previous investigations (12, 13, 17) includedEDTA in the tissue homogenization procedure, EDTA apparentlydoes not affect the density of plasma membranes. Because UDP-GlcNAc transferase and GDP-Man transferase activities areshifted to a lighter density after EDTA treatment while the K+-stimulated ATPase is not, these transferases are not associatedwith plasma membranes.

LINEAR SUCROSE DENSITY GRADIENT ANALYSES OF THE 250 TO13,000g PELLET

Glycoprotein glycosyl transferases have been reported in mito-chondria isolated from animal cells (3). Since considerable GDP-Man transferase and UDP-GlcNAc transferase activities were

pelleted between 250 and 13,000g (Table I), the distribution ofthese transferase activities was determined in sucrose gradients tosee if their activity was associated with subcellular componentsother than those found in gradients overlaid with microsomalpellets. GDP-Man transferase activities were detected at densitiesof 1.123 g/cc (corresponding to IDPase activity in Fig. 7), 1.148g/cc (corresponding to the Chl peak in Fig. 7), and 1.201 g/cc(unknown origin). The GDP-Man transferase activity associatedwith IDPase activity is identical to the previous results (Figs. I

FRACEIION NUMBER

FIG. 2. Sucrose density gradient distribution of particulate antimycinA-sensitive NADH-Cyt c reductase from pea cotyledons. A: the particulatefraction sedimented between 250 and 13,000g was split into equal halves.One half was washed in GM and repelleted at the initial force. NADH-Cyt c reductase was measured in the presence and absence of I ,UMantimycin A. B: the second half of the 250 to 13,000g fraction was washedin GM plus 5 mM EDTA and repelleted at the initial force. Assays werethe same as in A. C: the particulate fraction sedimented between 13,000and 40,000g was split into equal halves. One half was washed in GM andrepelleted at the initial force. Assays were the same as in A. D: the secondhalf of the 13,000 to 40,000g fraction was washed in GM plus 5 mmEDTA and repelieted at the initial force. Assays were the same as in A.All other gradient manipulations were the same as Figure 1.

Similar EDTA shifting experiments were performed to confirmthe localization of glycosyl transferase activities with ER. BothUDP-GlcNAc and GDP-Man transferase activities locatedaround 1.16 g/cc were shifted to a lighter density (1.10 g/cc) andtheir relocation was identical with the ER marker (Figs. 3 and4). In constrast, IDPase activity and its associated mannosyltransferase activity (1.123 g/cc) and the small amount of Chl didnot show a change in density (Figs. 3 and 4). A single peak ofChl (1.148 g/cc) was usually observed (Fig. 3) and the reason forthe appearance of a second small peak at 1.11 g/cc (Fig. 4) isunknown.The density of mannosyl transferase at 1.201 g/cc was not

affected by EDTA treatment (Fig. 4) although the magnitude ofactivity in the comparative gradients was different. This discrep-ancy may be caused by different divalent cation requirements ofdifferent mannosyl transferases and EDTA treatment may havelowered the endogenous ion level so that optimal concentrationwas not achieved in the standardized assay.

Glycosyl transferase activity has been reported to be associatedwith plasma membranes of animal cells (29). Since the reporteddensity range of plasma membranes isolated from plant tissue(1.16-1.18 g/cc) overlaps the density range of RER, the distribu-tion of plasma membranes in sucrose gradients was determined.

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FIG. 3. Sucrose density gradient distribution of particulate UDP-GlcNAc transferase isolated from pea cotyledons and sedimented between13,000 and 40,000g. A: one half of the particulate fraction sedimentedbetween 13,000 and 40,000g was treated as in Figure 2C and assayed forglycosyl transferase. B: the second half of the particulate fraction wastreated as in Figure 2D. All other gradient manipulations were the sameas Figure 1.

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MEMBRANE-BOUND GLYCOSYL TRANSFERASES

1.12gq,'cc 1.16,Icc 1.20q, 'cc 1. lOg/cc 1.12g,'cc 1. 20g,'cc

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mitochondria also have divalent cation-dependent phosphataseactivity.One peak of antimycin A-sensitive NADH-Cyt c reductase ac-

tivity was observed at a density (1.185 g/cc) typical for plantmitochondria (Fig. 2A). This observation confirms that the smallamount of antimycin A-sensitive activity observed at the samedensity in the previous gradients (Fig. 2, C and D) is associatedwith mitochondria. The protein peak at 1.185 g/cc also has anassociated pH 9 ATPase and Cyt c oxidase activity (inner mito-chondrial membrane marker) which are frequently used to identifymitochondria (Fig. 8A). Low levels of UDP-GlcNAc transferaseand GDP-Man transferase activities are found in the vicinity ofthe mitochondria but are not coincident with the protein peak(Fig. 6A).With EDTA treatment, the mitochondrial markers and the

associated antimycin A-sensitive NADH-Cyt c reductase activityare not shifted to a lighter density (Figs. 2A, 2B, 8A, and 8B).

1.12g/cc 1.15g/cc 1.20g/cc 1.10 1.12 1.15g/cc 1.20g/cc

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FRMCION NUMBER

FIG. 6. Sucrose density gradient distribution of particulate UDP-GlcNAc transferase and GDP-mannosyl transferase isolated from peacotyledons and sedimented between 250 and 13,000g. A: the normal treatedcontrol was prepared as in Figure 2A. B: the EDTA-treated fraction wasprepared as in Figure 2B. All other gradient manipulations were the sameas Figure 1.

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FiG. 5. Sucrose density gradient distribution of particulate ATPaseactivity isolated from pea cotyledons and sedimented between 13,000 and40,000g. Mg2+-ATPase activity was assayed at pH 6.5 in the presence andabsence of 50 mm KCI.

and 4). However, IDPase activity is also associated with the Chlpeak and the protein peak at a density of 1.185 g/cc. IDPaseactivity is associated with several membrane fractions and thisresult is consistent with the high proportion of this enzyme activityin the 250 to 13,000g pellet (Table I).

After EDTA treatment, the densities of GDP-Man transferaseactivities at 1.123 g/cc, 1.148 g/cc, and 1.201 g/cc are not changedalthough the magnitudes of activity are affected (Fig. 6, A andB). Only a very low level of GDP-Man transferase activity wasobserved at 1.148 g/cc. The activity at 1.201 g/cc was lower whencompared to the control (Fig. 6, A and B) and is consistent withprevious results (Fig. 4). The densities of IDPase activities didnot change but the magnitudes were affected, suggesting thatthese enzymes are divalent cation-dependent phosphatases. Thy-lakoid membranes have a Mg2e-dependent phosphatase (23) and

i0.83

FRACIION NUL8BER

FIG. 7. Sucrose density gradient distribution of particulate IDPase andChl isolated from pea cotyledons and sedimented between 250 and 13,000g.A: the normal treated control was prepared as in Figure 2A. B: theEDTA-treated fraction was prepared as in Figure 2B. All other gradientmanipulations were the same as Figure 1.

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FIG. 8. Sucrose density gradient distribution ofparticulate Cyt c oxidaseand pH 9 ATPase isolated from pea cotyledons and sedimented between250 and 13,000g. A: the normal treated control was prepared as in Figure2A. B: the EDTA-treated fraction was prepared as in Figure 2B. Allother gradient manipulations were the same as Figure 1.

However, a peak of NADH-Cyt c reductase activity insensitive toantimycin A appears at 1.102 g/cc (Fig. 2B). By shifting antimycinA-insensitive activity away from the mitochondrial region, anincreased inhibition of the mitochondrial associated enzyme bythe antibiotic is observed (Fig. 2, A and B). EDTA treatmentalso shifted GDP-Man transferase and UDP-GlcNAc transferaseactivities from the center of the gradient to a density of 1.102g/cc (Fig. 6, A and B). Unlike the GDP-Man transferase activity,all of the UDP-GlcNAc transferase activity has shifted to thelighter density indicating that this activity is specific for one typeof membrane. The concomitant relocation of both glycosyl trans-ferase activities with the ER marker indicates that these transferaseactivities found in the center of the gradient are associated witha small amount of RER sedimenting between 250 and 13,000g.

Developing peak cotyledons contain protein bodies which equi-librate at 1.253 g/cc in sucrose gradients (Fig. IOE) and further-more, the density of this organelle does not change after EDTAtreatment (Fig. 8). No glycosyl transferase activities were detectedwith the protein bodies (Fig. 6). The lack of coincidence betweenGDP-Man transferase and UDP-GlcNAc transferase activitieswith mitochondrial markers or protein bodies indicates that theglycosyl transferases pelleted between 250 and 13,000g (Table I)are not associated with these organelles.

ELECTRON MICROSCOPY

Gradients Overlaid with a 13,000 to 40,000g Pellet. To confirmthe reliability of the assays used to identify subcellular compo-nents, various particulate fractions collected from sucrose densitygradients were examined with an electron microscope. Electronmicrographs of intact tissue from 21-day-old cotyledons indicatedan abundance of RER (Fig. 9A). After tissue disruption, differ-ential centrifugation, and centrifugation in sucrose density gra-dients, particulate material collected at an average density of1.165 g/cc was identified morphologically as RER (Fig. 9, B andC). Membranes collected at an average density of 1.102 g/cc of agradient centrifuged with an "EDTA"-treated pellet containedvesicles which appeared to have remnants of ribosomes attached(Fig. 9D).

Electron micrographs of membranes collected at 1.123 g/ccwith the associated IDPase activity and mannosyl transferaseactivities showed fragments of Golgi apparatus and some largevesicles which have a double membrane (Fig. 9E). The lattervesicles may represent chloroplast envelopes which have beenreported at a similar density (23). It could not be ascertained

whether the IDPase activity and mannosyl transferase activitiesfound in this fraction are associated with Golgi apparatus mem-branes and/or chloroplast envelopes.

Gradients Overlaid with a 250 to 13,000g Pellet. The particulatefraction collected at a density of 1.123 g/cc of these gradientsalso had an associated IDPase and mannosyl transferase activities(Figs. 6A and 7A). Examination of this fraction showed thepresence of membranes similar to those obtained at the samedensity of a gradient centrifuged with a microsomal overlay(compare Figs. IOA and 9E). The major difference was the pres-ence of vesicles containing electron-dense particles (Fig. IOA).Examination of the Chl peak at 1.148 g/cc shows thylakoidmembranes from broken plastids (Fig. lOB). Cyt c oxidase, pH 9ATPase, and antimycin A-sensitive NADH-Cyt c reductase activ-ities located at a density of 1.185 g/cc are associated with mito-chondria (Fig. lOC). The fraction of unknown origin (1.201 g/cc)with an associated mannosyl transferase shows large sheets ofmembrane, large vesicles, and an occasional mitochondria (Fig.IOD). No enzyme activity was used to identify protein bodies,however electron micrographs of the fraction collected at 1.253g/cc show that this protein peak is composed mainly of proteinbodies (Fig. IOE).

INVOLVEMENT OF LIPIDS IN GLYCOSYL TRANSFER

To characterize the transfer of UDP-GlcNAc and GDP-Man,fractions isolated from a gradient overlaid with a microsomalpellet were combined around three specific regions. Fractionsbetween 1.11 and 1.13 g/cc were combined as were fractionsbetween 1.16 and 1.17 g/cc and between 1.19 and 1.21 g/cc.When these pooled fractions were analyzed for the involvementof lipid intermediates in the transferase reactions, it was foundthat glycosyl lipids were formed principally by membrane frac-tions with a density range of 1.16 to 1.17 g/cc (Table II). Thechloroform-methanol (2:1)-soluble material extracted from a par-ticulate fraction incubated with GDP-[14CJMan is mannosyl lipid(2) and the chloroform-methanol-water (1:1:0.3)-soluble com-pound is a lipid oligosaccharide which may contain both mannoseand N-acetylglucosamine (2). When UDP-[U14CJN-acetylgluco-samine is incorporated, the 2:1-soluble fraction contains N,N-diacetylchitibiosyl lipid and the 1:1:0.3-soluble component is alipid oligosaccharide (2).A small amount of radioactivity was transferred to the lipid-

free residue when UDP-["4CJGlcNAc was used as glycosyl donorand the most effective fraction was the RER (1.16-1.17 g/cc).Radioactivity accumulating in the residue is released by treatmentwith SDS (Table II) and protease (data not shown), indicatingthat GlcNAc becomes associated with glycoproteins. Table IIshows that the total radioactive accumulation in the lipid andresidue fractions approximated the radioactivity present in mem-brane fractions precipitated with trichloroacetic acid when UDP-['4CJGlcNAc was the glycosyl donor.

Considerable radioactivity accumulated in two residue fractionswhen GDP-["CJMan was used in the transferase assay (TableII). There was a major discrepancy between the trichloroaceticacid-precipitable radioactivity and radioactivity accumulated inthe lipid-soluble and residue fractions in transfer reactions me-diated by membranes with an average density of 1.123 g/cc (TableII). This may indicate the formation of water-soluble but trichlo-roacetic acid-insoluble oligomannans by these membranes. Thefraction of unknown origin (1.19-1.21 g/cc) showed a muchgreater transfer of radioactivity to the residue than to the lipidcomponents. Membranes with a density of 1.11 to 1.13 g/cctransferred more radioactivity to the residue fraction than to thechloroform-methanol-water (1:1:0.3)-soluble component. Very lit-tle radioactivity in these residues was released by SDS (Table II)and protease digestion for 80 hr did not release any label (datanot shown). Radioactivity in the residue obtained from RER was

456 Plant Physiol. Vol. 61, 1978

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Plant Physiol. Vol. 61, 1978 MEMBRANE-BOUND GLYCOSYL TRANSFERASES 457

-6~ ~ ~ ~ ~ -

14

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B w 0.5u *73

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FIG. 9. Electron microscopy of an intact pea cotyledon and membrane fractions collected from a sucrose gradient overlaid with a 13,000 to 40,OOOgpellet. A: a section of intact tissue from the second cell layer of a 21-day-old pea cotyledon. Note the abundance of RER. B: membranes representingthe density of 1.165 g/cc were repelleted from a gradient similar to Figure 2C and processed for electron microscopy. C: higher magnification of thefraction shown in B. D: membranes representing the density of 1.102 g/cc repelleted from a gradient similar to Figure 2D. E: membranes representingthe density of 1.123 g/cc repelleted from a gradient similar to Figure 2C.

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FIG. 10. Electron microscopy of membrane fractions collected from a sucrose density gradient overlayed with a 250 to 13,000g pellet. A-E:membrane fractions representing the following densities were repelleted from a gradient similar to Figure 2A. A: 1.123 g/cc; B: 1.148 g/cc; C: 1.185g/cc; D: 1.201 g/cc; E: 1.253 g/cc. All material was prepared for electron microscopy as described under "Materials and Methods." All sections werestained with uranyl acetate-lead citrate.

released by SDS treatment and protease digestion (data not ity during differential centrifugation indicates that this glycosylshown). transferase activity is associated with ER. In addition, both en-

CONCLUSIONS zyme activities are shifted to a lighter density (1.102 g/cc) insucrose gradients following treatment with EDTA (Figs. 2C, 2D,

Co-sedimentation of the majority of UDP-GlcNAc transferase and 3). This EDTA-induced change in density is caused by a

activity and antimycin A-insensitive NADH-Cyt c reductase activ- release of ribosomal subunits from RER with a concomitant

Plant Physiol. Vol. 61, 1978458

'4it iv

\1

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Plant Physiol. Vol. 61, 1978 MEMBRANE-BOUND GLYCOSYL TRANSFERASES 459

production of denuded or "smooth" ER (no other subcellularmembranes appear to be shifted by EDTA treatment). Morpho-logical examination of the membranes coliected around a densityof 1.165 g/cc primarily shows RER vesicles (Fig. 9, B and C)and after EDTA treatment, membranes shifting to a density of1.102 g/cc appear to be denuded ER (Fig. 9D).With UDP-['4C]GlcNAc as the substrate, the products of the

glycosyl transferase reactions in the ER are primarily lipid-linkedsugars (Table II) which have been implicated in the biosynthesisof glycoprotein in plants (2, 7, 8, 14) and animals (33). A lowlevel of incorporation into the lipid-free residue is observed.Radioactivity in the residue is released by solubilization in SDSand protease digestion indicating that counts are associated withglycoprotein. The observation that RER membranes can incor-porate radioactivity from UDP-['4CJGlcNAc into lipid interme-diates and glycoproteins indicates that glycoprotein assembly canoccur within this membrane system and furthermore that underour assay conditions UDP-GlcNAc transferase activity is specificfor RER in pea cotyledons.Mannosyl transferase activities are primarily associated with

three subcellular components. The enzyme activity located at1.123 g/cc (Figs. I and 4) coincides with a peak of IDPase activity.Morphological examination indicates a smooth membrane frac-tion (Fig. 9E) that contains fragments of Golgi apparatus andwhat appear to be plastid envelopes. Since plastid envelopes havebeen reported at a density of 1. I1 g/cc (23), the mannosyl trans-ferase and IDPase activity could not be conclusively associatedwith Golgi apparatus membranes. The mannosyl transferase ac-tivity which occurs in the center of the gradient (1.165 g/cc) iscoincident with the antimycin A-insensitive NADH-Cyt c reductaseactivity (Fig. 4A) and is considered to be associated with RER.The observation that both enzyme activities shift to a lighterdensity (1.102 g/cc) after EDTA treatment is consistent with thiscontention (Fig. 4).The mannosyl transferase activity at a density of 1.201 g/cc

appears in gradients overlaid with mitochondrial or microsomalpellets and is associated with a very low level of protein (Figs.4A and 6A). This transferase activity may be associated with amembrane stripped from an organelle during tissue homogeniza-tion. Morphology of the particulate material at 1.201 g/cc of agradient overlaid with a mitochondrial pellet shows large vesiclesand sheets of membrane (Fig. IOD). Merritt and Franke (20)have reported the presence of mannosyl transferase activity asso-ciated with nuclear membranes which have a reported density of1.21 g/cc for plants (22). The mannosyl transferase activity at1.201 g/cc may be associated with nuclear envelopes of thedeveloping pea cotyledon.Mannosyl transferase activities associated with RER synthesize

mannosyl lipids and lipid oligosaccharides which are involved inglycoprotein biosynthesis (2, 7, 8, 14). Since radioactivity incor-porated by the RER fraction can be released from the lipid-freeresidue by SDS treatment and protease, it appears that thismannosyl transferase activity associated with the ER functionsin glycoprotein biosynthesis. Since more radioactivity from GDP-[14CJMan is transferred to the residue fraction than to the lipidoligosaccharide by the transferases associated with membranescollected at a density of 1.123 g/cc and 1.201 g/cc, it appearsthat lipid intermediates are not involved in transfer of label tolipid-free residue. Radioactivity is not released from these residuesby treatment with SDS (Table II) or protease digestion andsuggests that mannose may be incorporated into polymannans(30).The analysis of sucrose density gradients with the crude mito-

chondrial overlay suggests that a low level ofmannosyl transferaseactivity is associated with the chloroplast thylakoid membranes.Low incorporation by this fraction has thus far precluded char-acterization of the product. The distribution of the major peaks

of GDP-mannosyl and UDP-GlcNAc transferases in these gra-dients is similar to the distribution of transferase activity ingradients overlaid with microsomal pellets although the magnitudeof enzyme activities is greater in the latter gradients. These trans-ferases are not associated with mitochondria or protein bodies.

Acknowledgments-The authors would like to thank J. Gaylor and M. A. Everett for providinguse of the electron microscope in the Department of Dermatology, University of OklahomaHealth Sciences Center.

LITERATURE CITED1. BASHA SMM, L BEEVERS 1976 Glycoprotein metabolism in the cotyledons of Pisum sativum

during development and germination. Plant Physiol 57: 93-972. BEEVERS L, RM MENSE 1977 Glycoprotein biosynthesis in cotyledons of Pisum sativum L.

Involvement of lipid-linked intermediates. Plant Physiol 60: 703-7083. BOSMANN HB, SS MARTIN 1969 Mitochondrial autonomy: incorporation of monosaccharides

into glycoprotein by isolated mitochondria. Science 164: 190-1924. BRETT CT, DH NORTHCOTE 1975 The formation of oligoglucans linked to lipid during

synthesis of fl-glucan by characterized membrane fractions isolated from peas. Biochem J148: 107-117

5. DEPIERRE JW, G DALLNER 1975 Structural aspects of the membrane of the endoplasmicreticulum. Biochim Biophys Acta 415: 411-472

6. DONALDSON RP, NE TOLBERT, C SCHNARRENBERGER 1972 A comparison of microbodymembranes with microsomes and mitochondria from plant and animal tissue. Arch BiochemBiophys 152: 199-215

7. ERICSON MC, DP DELMER 1977 Glycoprotein synthesis in plants. I. Role of lipid intermediates.Plant Physiol 59: 341-347

8. FORSEE WT, G VALKOVICH, AD ELBEIN 1976 Glycoprotein biosynthesis in plants. ArchBiochem Biophys 174: 469-479

9. GARDINER M, MJ CHRISPEELS 1975 Involvement of the Golgi apparatus in the synthesisand secretion of hydroxyproline-rich cell wall glycoproteins. Plant Physiol 55: 536-541

10. HARRIS PJ, DH NORTHCOTE 1971 Polysaccharide formation in plant Golgi bodies. Biochim.Biophys Acta 237: 5644

11. HoDGEs TK, RT LEONARD 1974 Purification of a plasma membrane-bound adenosinetriphosphatase from plant roots. Methods Enzymol 32: 392-406

12. HoDGEs TK, RT LEONARD, CE BRACKER, TW KEENAN 1972 Purification ofan ion stimulatedadenosine triphosphatase from plant roots: association with plasma membranes. Proc NatAcad Sci USA 69: 3307-3311

13. KOEHLER DE, RT LEONARD, WJ VAN DER WOUDE, AE LINKINs, LN LEWIS 1976 Associationof latent celulase activity with plasma membranes from kidney bean abscission zones.Plant Physiol 58: 324-330

14. LEHLE L, F FARTACZEK, W TANNER, H KAUSS 1976 Formation of polyprenol-linked mono-and oligosaccharides in Phaseolus aureus. Arch Biochem Biophys 175: 419-426

15. LEHLE L, W TANNER 1974 Membrane bound mannosyltransferase in yeast glycoproteinbiosynthesis. Biochim Biophys Acta 350: 225-235

16. LENNARZ WJ 1975 Lipid linked sugars in glycoprotein synthesis. Science 188: 986-99117. LEONARD RT, Wi VAN DER WOUDE 1976 Isolation of plasma membranes from corn roots

by sucrose density gradient centrifugation: an anomalous effect of Ficoll. Plant Physiol 57:105-114

18. LORD JM, T KAGAWA, TS MOORE, H BEEVERS 1973 Endoplasmic reticulum as the site oflecithin formation in castor bean endosperm. J Cell Biol 57: 659-667

19. LOWRY OH, NJ RoSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measurement with theFolin phenol reagent. J Biol Chem 193: 265-275

20. MERRITT WD, WW FRANKE 1976 Incorporation of galactose and mannose from sugar-nucleotides into endogenous acceptors of purified rat liver endomembranes. J CeU Biol 70:(Abstr Part 2) 304a

21. MOLNAR I 1975 A proposed pathway of plasma glycoprotein synthesis Mol Cell Biochem 6:3-14

22. PHILIPP El, WW FRANKE, TW KEENAN, I STADLER, ED IARASCH 1976 Characterization ofnuclear membranes and endoplasmic reticulum isolated from plant tissue. I Cell Biol 68:11-29

23. POINCELOT RP, PR DAY 1974 An improved method for the isolation of spinach chioroplastenvelope membranes. Plant Physiol 54: 780-783

24. POWELL IT, K BREW 1974 Glycosyltransferases in Golgi membranes of onion stem. BiochemJ 142: 203-209

25. RAY PM 1977 Auxin-binding sites of maize coleoptiles are localized on membranes of theendoplasmic reticulum. Plant Physiol 59: 594-599

26. RAY PM, TL SHININGER, MM RAY 1969 Isolation of beta-glucan synthetase particles fromplant cels and identification with Golgi membrane. Proc Nat Acad Sci USA 64: 605-612

27. REYNOLDS ES 1963 The use of lead citrate at high pH as an electron-opaque stain in electronmicroscopy. J Cel Biol 17: 208-212

28. SABATINI DD, Y TASHIRo, GE PALADE 1966 On the attachment of ribosomes to microsomalmembranes. J Mol Biol 19: 503-524

29. SHUR BD, S ROTH 1975 CeUl surface glycosyl transferases. Biochim Biophys Acta 415:473-51230. SMITH MM, M AXELOS, C P.AUD-LENOEL 1976 Biosynthesis of mannan and mannolipids

from GDP-Man by membrane fractions of sycamore ceUl cultures. Biochemie 58: 1195-121131. SPURR AR 1969 A low viscosity epoxy resin embedding medium for electron microscopy. J

Ultrastruct Res 26: 314332. VAN DER WOUDE WJ, CA LEMBI, DJ MORRE, JI KINDINGER, L ORDIN 1974 ,-Glucan

synthetases of plasma membrane and Golgi apparatus from onion stem. Plant Physiol 54:333-340

33. WAECHTER CJ, WJ LENNARZ 1976 The roe of polyprenol-linked sugars in glycoproteinsynthesis. Annu Rev Biochem 45: 95-112

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Subcellular Localization Glycosyl Transferases Involved in