mannose analog 1-deoxymannojirimycin inhibits … inhibits plant glycoprotein processing marked with...

Plant Physiol. (1989) 89, 1079-10840032-0889/89/89/1 079/06/$O1 .00/0

Received for publication August 15, 1988and in revised form November 8, 1988

Mannose Analog 1-Deoxymannojirimycin Inhibits the Golgi-Mediated Processing of Bean Storage Glycoproteins12

Alessandro Vitale*, Monica Zoppe, and Roberto BolliniIstituto Biosintesi Vegetali, Consiglio Nazionale delle Ricerche, via Bassini 15, 20133 Milano, Italy

ABSTRACT

The asparagine-linked oligosaccharide chains of glycoproteinscan be processed to form a wide variety of structures. The Golgicomplex is the main compartment involved in this processing. Inmammalian cells the first enzyme acting along the Golgi process-ing pathway is mannosidase 1, whose action is a prerequisite forany further processing and which is inhibited by the mannoseanalog 1-deoxymannojirimycin (dMM). To have insights into theprocessing pathway in plant cells, we have studied the In vivoeffect of dMM on the processing of the bean (Phaseolus vulgads)storage proteins phaseolin and phytohemagglutinin, two wellcharacterized plant glycoproteins. Cotyledons obtained fromdeveloping seeds were labeled with radioactive leucine, gluco-samine, or fucose in the presence or absence of dMM. Treatmentwith dMM fully inhibited the acquisition of resistance to endo-#-N-acetylglucosaminidase H by phaseolin and phytohemagglutininand the incorporation of fucose into protein. Furthermore, theapparent molecular weight of the polypeptides of phaseolin andphytohemagglutinin synthesized in dMM-treated cotyledons wasconsistent with the exclusive presence of oligommanose oligo-saccharide chains which had not been processed in the Golgicomplex. The inhibition of processing did not prevent exit fromthe Golgi complex, and most probably the storage proteins werecorrecty targeted to the protein bodies as indicated by the post-translatonal polypeptide cleavage of phaseolin. These resuitsindicate that the action of a mannosidase is the first obligatorystep of Golgi-mediated processing also in a plant cell and, to-gether with data obtained in other laboratories on the In vitrospecificity of glycosidases and glycosyltransferases present inthe Golgi complex of plant cells, support the hypothesis that thekey early reactions in Golgi-mediated processing are similar ifnot identical in plants and mammals.

The proteins that are synthesized on the ER of eukaryoticcells can be cotranslationally glycosylated at Asn residuespresent in the sequence Asn-X-Ser/Thr. The reaction consistsof the transfer of an oligosaccharide chain (OS3) having thestructure Glc3Man9GlcNac2 from its dolichol derivative to theAsn in the nascent glycoprotein. This oligosaccharide is thenprocessed in a concerted, stepwise set of reactions catalyzedby several enzymes (reviewed in Ref. 18). The processing

'Supported in part by the Progetto Strategico Agrotecnologie ofthe Consiglio Nazionale delle Ricerche.

2Dedicated to the late Giuseppe Torti.3Abbreviations: OS, oligosaccharide chain; dMM, 1-deoxyman-

nojirimycin; endo H, endo-fl-N-acetylglucosaminidase H; PHSL,phaseolin; PHA, phytohemagglutinin.

pathway has branching points, some of which are probablycell type-specific. Furthermore, structures in which processinghas been interrupted virtually at any step along the pathwayhave been found; these characteristics of the pathway thusmake possible the formation of many different Asn-linkedOS. Glycosylation changes the molecular characteristics ofthe proteins and this can influence protein stability, biologicalproperties, and destiny (21).The processing pathway has been most studied in mam-

malian cells: processing starts in the ER where the three Glcresidues are removed by the sequential action of glucosidaseI and II and one to three of the Man residues can be removedby an a-1,2 mannosidase. Glucosidase I and II have beenidentified also in plant cells (27), while it is not yet knownwhether an ER mannosidase participates in glycoprotein proc-essing in plants. Further processing requires transport to theGolgi complex, where most of the processing enzymes arelocated. The first enzyme acting in the Golgi complex inmammalian cells is the Golgi a-1,2-mannosidase (mannosi-dase I), which yields the structure Man5GlcNAc2: this is thesubstrate for GlcNAc transferase I which produces theGlcNAcMan5GlcNAc2 structure, the most important 'cross-road' of processing (18). The mannose analog dMM inhibitsmannosidase I in mammalian cells and glycoproteins synthe-sized in vivo in the presence ofdMM have only OSs with thestructure Man6_9GlcNAc2, indicating that the inhibition ofmannosidase I blocks any further processing (1, 2, 12, 13).

In vitro studies using a Golgi-enriched fraction from devel-oping cotyledons of the common bean, Phaseolus vulgaris,and different glycopeptide acceptors revealed the presence ofGolgi transferases with substrate specificities analogous tothose of the enzymes present in mammalian cells (17). Also,an a-mannosidase activity has been purified from the micro-somal fraction of mung bean seedlings; in vitro experimentsshowed that this enzyme has substrate specificity similar tothat of mammalian Golgi mannosidase I and is inhibited bydMM (26). However there are no in vivo data about theinvolvement of such an enzyme in glycoprotein processing inplant cells. Being interested in the regulation of processing ofplant glycoproteins, we decided to study the effect of dMMon the synthesis of PHSL and PHA, two well-characterizedglycoproteins, in bean cotyledons. PHSL and PHA are themajor storage proteins of the common bean. They are syn-thesized on the ER and transported via the Golgi apparatusto the protein bodies, where they accumulate (reviewed inRef. 8). During synthesis and intracellular transport theseproteins undergo a series of co- and post-translational proc-essing events which include N-glycosylation and processing of

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Plant Physiol. Vol. 89, 1989

the OS (24, 31). Our results are consistent with the fullinhibition of Golgi-mediated processing by dMM. PHSL andPHA carrying unprocessed OS exit from the Golgi complexand most probably are targeted to their normal destination,the protein bodies.

MATERIALS AND METHODS

Materials

Plants ofPhaseolus vulgaris cv Greensleeves were grown ina growth chamber with a 14 h photoperiod, day at 25°C, nightat 20°C. Chemicals were purchased from Carlo Erba, Milano,unless otherwise indicated, and radiochemicals from Amer-sham International. Endo H from Streptomyces plicatus anddMM were purchased from Miles Laboratories.

Radioactive Labeling

Excised mid-maturation cotyledons were placed, flat sidedown, on 20 ,uL of 5 mM dMM (dissolved in water) or 20 ,Lof water (for the control) and incubated for 150 min at roomtemperature. After this pretreatment, the 20 ,uL were replacedwith again 20 ,uL of the same solution containing 370 kBq ofone of the following radioactive precursors: L-[5,6-3H]leucine(6.77 TBq/mmol), D-[6-3H]glucosamine hydrochloride (1.48TBq/mmol), L-[5,6-3H]fucose (1.67 TBq/mmol). For chaseexperiments, cotyledons were rinsed in water after the labelingand then placed on 20 ,uL of nutrient medium (3) which, fordMM treatment, was supplemented with 5 mM dMM.

RESULTS

dMM Does Not Affect Storage Protein Synthesis butAlters ProcessingTo study the effect of dMM on protein synthesis and

processing, developing cotyledons were incubated for 150 minin the presence of 5 mm dMM or in water as a control, thenpulse labeled for 3 h with [3H]leucine and chased for 21 h toallow accumulation of radioactive protein in the proteinbodies. Cotyledons treated with the inhibitor were maintainedin 5 mM dMM throughout the 24 h pulse-chase. Proteinspresent in the soluble fraction and in the total membranefraction were then analyzed by SDS-PAGE and fluorography(Fig. 1). Storage proteins contained in the protein bodies,which break during homogenization (5), are recovered withthe soluble fraction. The precursors of storage proteins, asso-ciated with the ER and the Golgi complex, are instead re-covered with the membrane fraction. Treatment with dMMdid not inhibit protein synthesis, as judged by the amount of[3H]leucine incorporated into proteins (compare lanes 1 and3 with lanes 2 and 4, respectively, in Fig. 1), and did not affectthe exit of storage proteins from the endomembrane systemconstituted by the ER and Golgi. In fact, at the end of thechase, most of the PHSL and PHA was recovered with thesoluble fraction both in dMM-treated and untreated cotyle-dons (lanes 3 and 4). It was evident however that dMM causedthe accumulation of PHSL and PHA polypeptides with in-creased apparent mol wt compared to the control (the posi-tions of the PHSL and PHA polypeptides in control are

dMMII rl. FTI- II

Homogenization, Cell Fractionation and Isolation of PHAand PHSL

The top half of the cotyledons was discarded and theremainder was homogenized in a mortar with 0.5 mL/coty-ledon of 100 mM Tris-Cl, 1 mM EDTA, pH 7.8 (buffer A),containing 12% (w/w) sucrose. Both the mortar and thehomogenization solution were ice-cold. After centrifugationfor 5 min at 10OOg at 4°C, the supernatant was layered overa dicontinuous sucrose gradient constituted by 6 mL ofbufferA containing 16% (w/w) sucrose on top of 4 mL of buffer Acontaining 54% (w/w) sucrose. Centrifugation was for 90 minat 35,000 rpm at 4°C in a Beckman SW40 rotor. The loadportion of the gradient (soluble fraction) and the organelleswhich sedimented on top ofthe 54% sucrose layer (membranefraction) were then recovered. The soluble fraction containedPHA and PHSL released from the protein bodies, which hadbeen disrupted during homogenization (5); the membranefraction contained PHA and PHSL precursors present in theER and in the Golgi complex. The two storage proteins wereisolated as described (6), using monospecific antibodies co-valently bound to Sepharose 4B (Pharmacia). Either totalproteins present in each subcellular fraction or immunoiso-lated proteins were analyzed by SDS-PAGE and fluorographyas described elsewhere (4). Digestion ofPHA and PHSL withendo H was done as described by Vitale et al. (32).

,5 6 7 8

Figure 1. Analysis by SDS-PAGE and fluorography of total proteinsand PHA synthesized in the presence of dMM. Labeling was with[3H]leucine (3 h pulse, 21 h chase). After homogenization, membrane(m) and soluble (s) subcellular fractions were isolated and analyzed.For total protein analysis (lanes 1-4), material from 1/30 of cotyledon(lanes 1 and 2) and 1/175 of cotyledon (lanes 3 and 4) was loadedon the gel. For comparison of PHA pattems (lanes 5-8), immunopre-cipitated PHA with a radiactivity of approximately 3000 cpm wasloaded on each lane. The positions of the polypeptides of PHSL andPHA in lane 4 are indicated by solid circles and asterisks, respectively.

1 080 VITALE ET AL.

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1-DEOXYMANNOJIRIMYCIN INHIBITS PLANT GLYCOPROTEIN PROCESSING

marked with solid circles and asterisks, respectively). Thisindicates that processing of both proteins is altered in thepresence of the inhibitor.

PHA Synthesized in the Presence of dMM Does NotChange Electrophoretic Mobility during IntracellularTransport and Does Not Acquire Resistance to Endo HDigestion

PHA is a tetramer composed of two similar subunits withmol wt around 34,000 and 32,000 (19) (they are marked withasterisks in lane 4 of Fig. 1) which are synthesized on the ERand undergo virtually identical processing (25, 30, 31). Co-translationally a signal peptide is cleaved and two oligoman-nose OS are added. During passage ofPHA through the Golgicomplex the OS distal to the N terminus is processed: 4 to 6mannose residues are removed and one residue of fucose andxylose added as well as probably two terminal N-acetylglucos-amine residues. The terminal N-acetylglucosamine residuesare removed after PHA has reached the protein bodies. Theother OS is almost completely unprocessed and has a finalstructure Man8 9GlcNAc2. PHA was immunoprecipitatedfrom the preparations shown in lanes 1 to 4 of Figure 1 (theimmunoprecipitates are in lanes 5-8 of Fig. 1; most of PHAwas recovered with the soluble fractions, but equal amountsof radioactivity were loaded in each lane to facilitate compar-isons of the polypeptide patterns). A small amount of labeledPHA was recovered from the membrane fractions even afterthe long chase: in untreated cotyledons this PHA had a ratherdiffuse three-banded pattern in which the central band wasthe most intense (Fig. 1, lane 7). This is because the membranefraction contains both the ER and the Golgi complex: proc-essing of the OS causes a decrease in the apparent mol wt ofPHA polypeptides (this can be seen by comparing the patternsof glucosamine and fucose labeled PHA present in the mem-brane fraction of pulse-labeled cotyledons; see lanes 5-7 inFig. 3) and therefore the pattern observed is the result ofoverlapping of polypeptides with unprocessed OS (presentmainly in the ER) and processed OS (present in the Golgicomplex). Mature PHA recovered with the soluble fraction(Fig. 1, lane 8) showed the two typical bands, with mobilitieswhich do not exactly correspond with any ofthe componentspresent in the membrane fraction: this is due to removal ofthe terminal N-acetylglucosamine residues, which further in-creases the mobility of PHA polypeptides (30, 31). A com-pletely different situation was observed in dMM-treated co-tyledons: PHA from the membrane and the soluble fractions(lanes 5 and 6 in Fig. 1) had identical patterns, representedby two well-resolved bands. The electrophoretic mobilitieswere also identical to those of radioactive PHA polypeptidespresent after a short pulse in the membrane fraction ofuntreated cotyledons (not shown).These results suggest that dMM blocked the processing of

the OS distal from the N terminus. This hypothesis was firsttested by examining PHA digested with endo H. Processingin the Golgi complex leads to acquisition ofendo H resistance(31). Therefore, endo H digestion of PHA that accumulatesin normal conditions leads to the removal of only one OSfrom each polypeptide (Fig. 2, lanes 2 and 4). When PHAisolated from the soluble fraction of dMM-treated cotyledons

endo H:

dMM:

_-

+ +

+ - +

1 2 3 4 5 6

3 3

Figure 2. Endo H treatment of PHA. Lanes 2 to 5, PHA was isolatedfrom the soluble subcellular fraction of developing cotyledons whichhad been labeled with [3HJleucine (3 h pulse, 21 h chase), in theabsence or the presence of dMM; the protein was denatured byboiling and incubated as described elsewhere (32) without or withendo H. Lanes 1 and 6, proteins from the membrane subcellularfraction of cotyledons labeled for 2 h with [3H]leucine in the presenceof tunicamycin; arrowheads indicate unglycosylated PHA polypep-tides in lane 6. Analysis was by SDS-PAGE and fluorography.

was digested with endo H (Fig. 2, lanes 3 and 5), the resultingpolypeptides had electrophoretic mobilities corresponding tothose of unglycosylated PHA made in the presence of tuni-camycin (Fig. 2, arrowheads in lane 6), indicating removal ofboth OS. Therefore dMM treatment inhibits acquisition ofendo H resistance.

dMM Blocks Incorporation of Fucose

When bean cotyledons are incubated in the presence of[3H]fucose, the radioactive sugar is readily incorporated intoPHA as a result of the Golgi mediated processing (7). Coty-ledons, treated with dMM or untreated controls, were labeledfor 2 h either with [3Hjglucosamine or with [3H]fucose andthe membrane fraction isolated. This fraction contained mostof the radioactive PHSL and PHA, which had not yet beentransported to the protein bodies. Figure 3 shows the totalradioactive proteins present in the membrane fractions (lanes1-4) and immunoisolated PHA (lanes 5-8). PHSL and PHAare the most abundant glycoproteins synthesized in develop-ing bean cotyledons and therefore incorporate most of the[3H]glucosamine, both in control and in dMM treated coty-ledons. PHA incorporated most of the [3H]fucose in thecontrol (PHSL does not contain fucose), but fucose incorpo-ration was almost completely inhibited by dMM, both inPHA and in other minor glycoproteins. Also, the pattern ofglucosamine-labeled PHA was altered by dMM consistentlywith the above reported results: two defined bands could be

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Plant Physiol. Vol. 89, 1989

>- uc. GICN_ M_ - _ _

dMM 4

1 2 3 4

GIcN ,IB.57..

5 6 7 8

i4.

Figure 3. The effect of dMM on glucosamine and fucose incorpora-

tion. Developing cotyledons were labeled for 2 h with either [3HI

glucosamine or [3H]fucose, in the presence or in the absence of dMM,

and the membrane subcellular fraction was isolated. Lanes 1 to 4,

total proteins from the membrane fraction; lanes 5 to 8, immunopre-

cipitated PHA. Analysis was by SDS-PAGE and fluorography. Materialfrom equivalent amounts of tissue extract was loaded in each lane.

The positions of the polypeptides of PHSL and PHA in lane 4 are

indicated by solid circles and asterisks, respectively.

detected instead of the smeared pattern in the control (com-

pare lanes 5 and 6 in Fig. 3).

PHSL was immunoprecipitated from the soluble fractionsof the [3H]leucine pulse-chased cotyledons shown in Figure 1(Fig. 4). As shown by the total protein patterns (Fig. 1), dMMcaused the accumulation of PHSL polypeptides with de-creased mobilities with respect to normal PHSL (comparelanes 1 and 2 in Fig. 4). The polypeptides accumulated in thepresence ofdMM had a mobility higher than the polypeptidesof the PHSL precursor isolated from the membrane fractionof control cotyledons pulse-labeled for 3 h with [3H]leucine(lane 3 in Fig. 4). We interpret this to indicate that dMMcaused the accumulation of PHSL which was not processedon the OS, but underwent post-translational removal of theshort amino acid sequence because it reached the proteinbodies. This hypothesis was tested by endo H digestion. Lane4 in Figure 4 shows the pattern of PHSL isolated from thesoluble fraction of control cotyledons and then digested withendo H. Deglycosylated polypeptides (arrowheads) and poly-peptides with one OS (above each deglycosylated polypeptide)are produced, in a ratio of about 1:1. In PHSL a' and ," aremuch more abundant than a' and P3': our results thereforeindicate that normally also some ofthe a" and (i" polypeptideshave one processed OS, which is resistant to endoH digestion.More than 95% of PHSL accumulated in dMM-treated co-tyledons was totally susceptible to endo H (lane 5 in Fig. 4),and deglycosylated polypeptides produced after digestion wereindistinguishible in size from those obtained from normalPHSL (compare polypeptides marked by arrowheads in lanes4 and 5 of Fig. 4). Digestion of PHSL precursor present inthe membrane fraction ofuntreated cotyledons (lane 6 in Fig.4) produced deglycosylated polypeptides (more abundant)and once glycosylated polypeptides (less abundant). This isthe result expected from digestion of the mixture of PHSLprecursors present in the ER and in the Golgi complex. Also,deglycosylated polypeptides (arrowheads in lane 6) had mo-

dMM Inhibits Processing of the OS of PHSL, but Not ItsPolypeptide Cleavage in the Protein Bodies

PHSL is a trimeric protein which, at the polypeptide level,is composed by two size classes, a and ,B, of very similarpolypeptides (14, 23). Each polypeptide has two potential N-glycosylation sites and during synthesis on the ER the signalpeptide is removed and each polypeptide is glycosylated witheither one (a' and ft') or two (a" and fl") OS: this gives riseto a four-banded electrophoretic pattern on SDS-PAGE (6).The processing and the final structure of the OS of PHSLhave recently been the subject ofan extensive study (24). TheOS of a" and fl' do not appear to be processed during passagethrough the Golgi complex; however, when only the first Asnis glycosylated, as in a' and fl', the OS undergoes processingevents very similar to those that occur on PHA. The onlydifference between PHA and PHSL is the absence of fucosy-lation on PHSL. In the protein bodies the polypeptides ofPHSL also undergo the removal of a short amino acid se-quence (5, 29); only a few amino acids are removed, but theexact site of cleavage has not yet been determined. Processingevents occurring in the Golgi complex and in the proteinbodies cause an increase in the electrophoretic mobility ofphaseolin polypeptides, more pronounced in the case of a'and ,B', than of a" and f,".

J.MM

1 2 3

. -.

4 5 6 7 8 9

..£..._

Figure 4. The effect of dMM on the processing of PHSL. Phaseolinwas immunoisolated either from the soluble fraction (s) of developingcotyledons labeled with [3H]leucine (pulse 3 h, chase 21 h) in thepresence or the absence of dMM, or from the membrane fraction (m)of developing cotyledons pulse-labeled for 3 h with [3H]leucine in theabsence of dMM. The immunoprecipitates were analyzed by SDS-PAGE and fluorography either without further treatment (lanes 1-3)or after incubation in the presence or in the absence of endo H (lanes4-9). Arrowheads indicate fully deglycosylated polypeptides.

1 082 VITALE ET AL.



1-DEOXYMANNOJIRIMYCIN INHIBITS PLANT GLYCOPROTEIN PROCESSING

bilities lower than those of the correspondent polypeptidespresent in the soluble fraction in both untreated and dMM-treated cotyledons. The difference in electrophoretic mobilitycorresponded to that which can be observed between theprecursor and mature forms of unglycosylated PHSL synthe-sized in tunicamycin-treated cotyledons (not shown). Weinterpret the results of endo H digestion to confirm ourhypothesis that dMM inhibited processing of the OS of phas-eolin but not transport to the protein bodies, where post-translational cleavage of the polypeptide chain occurs.

DISCUSSION

In mammalian cells Golgi mediated processing starts withthe action of mannosidase I, which removes a-1,2-linkedmannose residues, allowing the subsequent reactions to occur.The aim of this work was to test whether the removal of a-1,2-linked mannoses from the oligomannose OS of glycopro-teins is a prerequisite for the following Golgi-mediated proc-essing reactions also in plant cells. For this purpose we haveused the mannose analog dMM, which has been shown toinhibit mannosidase I in intact mammalian cells (2, 12, 13).Recent work from our laboratory indicated that acquisitionof endo H resistance by a glycoprotein synthesized uponmRNA injection in oocytes ofXenopus laevis is fully inhibitedby dMM, suggesting that the important role played by man-nosidase I is not limited to mammalian cells (1 1).We have shown here that in the presence of dMM, PHA

and PHSL do not acquire endo H resistance. Furthermore,PHA does not incorporate fucose. We assume that also incor-poration of xylose is inhibited by dMM, both in PHA andPHSL, since xylose attachment confers endo H resistance tothe OS (24). Finally, the electrophoretic mobilities of thepolypeptides of the two storage proteins synthesized in thepresence ofdMM are consistent with full inhibition of Golgi-mediated processing. We therefore propose that an enzymeanalogous to mammalian mannosidase I is inhibited bydMMand that this causes a block in the processing of N-linked OSwhich occurs in the Golgi complex ofbean cotyledonary cells.The block obtained using dMM is different from the oneobtained in the same cells due to the action of anothermannosidase inhibitor, swainsonine. In mammalian cells,swainsonine inhibits mannosidase II, the enzyme which re-moves a-1,3- and a-1,6-linked mannoses thus convertingGlcNAcMan5GlcNAc2 to GlcNAcMan3GlcNAc2 (28). It hasbeen shown that swainsonine most probably has the sameeffect on the processed OS ofPHA and that, as in mammaliancells (20), the inhibitor does not prevent fucosylation in plantcells (10, 15). Therefore, in bean cotyledonary cells two dif-ferent mannosidases are acting on glycoproteins, apparentlywith the same specificities of mammalian mannosidase I andII. Our results obtained in vivo confirm and extend the workof other investigators who studied, in vitro, the substratespecificities of OS processing enzymes from bean cotyledons(17). On the whole these studies strongly suggest that thereactions until the formation of GlcNAc2Man3GlcNAc2 arethe same in plant and mammalian cells. Whether the subse-quent reactions, which in mammalian cells occur mainly inthe medial and trans cisternae of the Golgi complex (18),occur along a similar pathway in the plant cells remains to be

determined. The fact that addition of sialic acid has neverbeen observed in plant glycoproteins may indicate that unlikethe key early reactions, late reactions may be not widelyconserved through evolution; late reactions appear to bespecific for different animal cell types ( 16, 22).The storage proteins of bean cotyledons accumulate in the

vacuolar protein bodies. Previous work from our laboratoryhas determined that glycosylation is not necessary for target-ing of PHA to the protein bodies (3). We have also shownthat the post-translational processing ofthe polypeptide chainsof PHSL can occur in the absence of OS (29); this processingtakes place in the protein bodies and therefore, also for PHSL,glycosylation is not a prerequisite for correct intracellulartargeting. Radioactive PHSL and PHA that accumulate aftera pulse-chase labeling in the presence of dMM can be re-covered with the soluble fraction which contains the proteinsreleased from the protein bodies broken during homogeniza-tion. Furthermore, the polypeptide chains of this form ofPHSL are post-translationally processed. These data stronglysuggest that also abnormal glycosylation, due to the inhibitionof processing of the OS that are normally destined to beextensively modified, does not interfere markedly with theintracellular transport of bean storage proteins.

ACKNOWLEDGMENT

We are grateful to Aldo Ceriotti for critical reading of the manu-script.

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30. Vitale A, Ceriotti A, Bollini R, Chrispeels MJ (1984) Biosyn-thesis and processing of phytohemagglutinin in developingbean cotyledons. Eur J Biochem 141: 97-104

31. Vitale A, Chrispeels MJ (1984) Transient N-acetylglucosaminein the biosynthesis of phytohemagglutinin: attachment in theGolgi apparatus and removal in protein bodies. J Cell Biol 99:133-140

32. Vitale A, Sturm A, Bollini R (1986) Regulation of processing ofa plant glycoprotein in the Golgi complex: a comparative studyusing Xenopus oocytes. Planta 169: 108-116

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mannose analog 1-deoxymannojirimycin inhibits … inhibits plant glycoprotein processing marked with...

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