Electron microscope localization of concanavalin a-binding sites in plasma membrane- and endoplasmic reticulum-rich fractions from maize coleoptiles

Plant Science Letters, 30 (1983) 239--248 Elsevier Scientific Publishers Ireland Ltd.

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ELECTRON MICROSCOPE LOCALIZATION OF CONCANAVALIN A-BINDING SITES IN PLASMA MEMBRANE- A N D ENDOPLASMIC RETICULUM-RICH FRACTIONS FROM MAIZE COLEOPTILES M.A. H A R T M A N N ' ,

P. B E N V E N I S T E a and J.C. R O L A N D b

aE.R.A, au C.N.R.S. No. 487 (Structure et fonction des membranes p#}ricellularies, ContrSle de la biosynthdses des stdrols),Institutde Botanique, 28 rue Goethe, 67083 Strasbourg Cddex and bLaboratoire de Biologie Vdgdtale, Ecole Normale Supdrieure, 24 rue Lhomond, 75231 Pads Cddex 05 (France)

(Received September 30th, 1982) (Revision received November 16th, 1982) (Accepted December 6th, 1982)

SUMMARY

The binding of concanavalin A (Con A) to plasma membrane(PM)- and endoplasmic reticulum(ER)-rich fractions from etiolated maize coleoptiles has been investigated using sequential treatment with Con A and mannosylferritin (Man-Fer). PM vesicles were shown to be extensively labelled at their outer surface, indicating the presence of available mannosyl and/or glucosyl residues in this fraction. Ferritin particles presented a clustered distribution. No label was observed on control vesicles stained in the presence of a-methyl-D-mannoside (aMM). Membranes of the ER-rich fraction appeared largely devoid of ferritin particles. Combining this electron microscope study with procedures which render the interior of the vesicles accessible to the lectin, it was found that whereas no modification in the label of PM vesicles occurred, ER membranes were extensively labelled on their cisternal face. The results are totally in agreement with biochemical data reported in the preceding paper (Hartmann et al., Plant Sci. Lett., 30 (1983) 227) and suggest that PM and ER vesicles retain their orientation after grinding of coleoptile tissue.

Key words: Plasma membrane -- Endoplasmic reticulum -- Concanavalin A binding -- Mannosyl-ferretin -- Electron microscopy

Abbreviations: Con A, Concanavalin A; ER, endoplasmic reticulum;Man-Fer, mannosylferritin; aMM, a-methyl-D-mannoside; PACP, phnsphotungstic acid-chromic acid procedure; PM, plasma membrane; UDPase, uridine 5'-diphosphatase. 0304-4211/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

240 INTRODUCTION

Electron-microscopic markers, especially ferfitin-conjugated lectins, have turned out to be useful in the localization of carbohydrate residues on membrane surfaces [1]. Evidence for Con A binding sites at the surface of protoplasts from several plant sources has been presented [2--6]. However, the capacity for cell wall regeneration, a process which may be very rapid [7], by isolated protoplasts has made lectin binding to nascent cell wall material rich in carbohydrates difficult to distinguish from its binding to the PM. These difficulties have been circumvented with the use of isolated PM-rich preparations and such studies have provided evidence of Con A binding sites on PM membranes [8]. Regarding the precise orientation of carbohydrate residues on intracellular membranes from plant cells, in particular cytoplasmic versus luminal exposure of these compounds, little work has yet been carried out. The present study is concerned with establishing evidence for Con A binding sites in PM- and ER-rich fractions from etiolated maize coleoptiles using a two-step labelling method with Con A and Man-Fer [9]. Con A-ferritin binding was tested with the combination of the phosphotungstic acid-chromic acid procedure (PACP) staining. The levels of Con A binding by PM and ER vesicles before and after treatment of both preparations with 0.02% Triton X-100 or acid pH, procedures which are known to disrupt membrane integrity [10,11], are investigated. The results obtained by this cytochemical method are compared with quantitative biochemical data reported in the preceding paper [12]. MATERIALS AND METHODS

Plant material Maize seeds (Zea mays cv. LG-11) were allowed to germinate in the dark at 25°C. The coleoptiles were excised after 6 days.

Isolation o f membrane fractions PM- and ER-rich fractions were isolated as previously described [13]. Both fractions were washed free of sucrose by repeated centrifugation and then resuspended in 0.02 M Tris--HC1 (pH 7.5) containing 0.2 M NaC1 (Buffer A). Binding o f Con A to membrane fractions Binding experiments were carried out by incubating membrane fractions (0.8--1.2 mg of protein) at room temperature for 45 rain with 6.4 #M Con A in buffer A in a total volume of 2 ml. Control experiments were performed with 0.1 M aMM in addition to the above constituents. Samples were then centrifuged at 56 000 × g for 30 rain. Pellets were resuspended in 1 ml of buffer A containing or not 0.1 M aMM.

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Ferritin labelling After washing twice by centrifugation under the same above conditions to remove unbound Con A, pellets were resuspended into 1 ml of Man-Fer at an approximate protein concentration of 0.2 mg/ml in buffer A. Suspensions were kept at room temperature for 45 rain, then centrifuged at 56 000 × g for 30 min. Electron microscopy After ferritin labelhng, membrane fractions were fixed in 3.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) at 4°C for 1 h. After three washes of 1 h each with phosphate buffer, samples were postfixed in buffered 1% OsO4 at 4°C for 1 h. Then, they were rinsed in phosphate buffer, dehydrated with graded solutions of ethanol and embedded in an araldite-epon mixture (4:5, v/v). After being cured, specimens were sectioned. Thin sections were stained with either uranyl acetate and lead citrate or PACP [14] and examined in a Philips electron microscope at 80 kV. Effect o f Triton X-1 O0 and acid pH on Con A binding by PM and ER vesicles Membrane fractions were treated at 30°C for 15 min with 0.01--0.02% (w/v) Triton X-100 in 0.1 M Tris=-HC1 (pH 8.0) or at room temperature for 30 rain in 0.05 M sodium acetate-acetic acid (pH 5.0) in a total volume of 8 ml. In both cases, 1 mM phenyl methyl sulfone fluoride was added in the incubation medium. Control experiments were performed by incubating membrane preparations in 0.05 M Tris---HC1 (pH 8.0) at room temperature for 30 min. Samples were then centrifuged at 110 000 × g for 40 min. Pellets were resuspended in 2 ml of buffer A and tested for Con A-ferritin labelling and UDPase activity. UDPase assays They were performed as indicated in Ref. 15. RESULTS

Con A-ferritin labelling o f PM and ER vesicles As previously reported [16], the PM-rich preparation from etiolated maize coleoptiles consists of large smooth vesicles (average diameter of 0.3/JM), with no identifiable membrane sheets. Most of the vesicles are contrasted by PACP, proving thus a high level of enrichment of this fraction with PM [17]. The specificity of this staining procedure for PM from maize was checked with whole tissues [16]. The absence of contamination originating from endomembranes, especially ER membranes, is attested by the use of appropriate enzymic markers [13]. PM vesicles were sequentially treated with Con A ahd Man-Fer. First, PM vesicles were incubated with Con A under conditions similar to those employed in the radioactive lectin-binding assays [12]. Then, they were

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Figs. 1 and 2. Low magnifications of pellets of fractions enriched in plasma membranes and incubated with Con A-ferritin at pH 8 (Fig. 1: x 40 000, Fig. 2: x 67 000) uranyl acetate-lead citrate staining. w a s h e d a n d i n c u b a t e d w i t h M a n - F e r . As s h o w n in Figs. 1 - - 3 , fen~tin particles w e r e c o n f i n e d t o t h e e x t e r n a l s u r f a c e o f PM vesicles w i t h n o n e f o u n d o n t h e i n t e r n a l face or in t h e i n t e r i o r o f t h e vesicles. T h e d i s t r i b u t i o n o f particles a p p e a r s s o m e w h a t p a t c h y , w i t h clusters l o c a t e d in areas n e a r vesicle c o n t a c t s . E l e c t r o n m i c r o g r a p h s o f P A C P stained sections s h o w e d ferritin particles t o b e a s s o c i a t e d o n l y w i t h t h e P A C P p o s i t i v e l y stained vesicles (Fig. 3). C o n t r o l e x p e r i m e n t s r u n w i t h 0.1 M a M M y i e l d e d vesicles clean o f ferritin particles (Fig. 4), p r o v i d i n g e v i d e n c e t h a t t h e labelling was specific f o r C o n A. T h e E R - r i c h f r a c t i o n is c o n s t i t u t e d o f vesicles w h i c h are smaller t h a n PM-vesicles a n d n o t reactive t o PACP [ 1 6 ] . A f t e r i n c u b a t i o n w i t h C o n A a n d M a n - F e r , o n l y a l o w labelling o f E t t vesicles was Observed (Fig. 7). F o r p o s i t i v e l y labelled vesicles, ferritin particles w e r e f o u n d t o b e a s s o c i a t e d w i t h t h e o u t e r face o f t h e vesicles. W h e n labelling e x p e r i m e n t s w e r e p e r f o r m e d

Figs. 3--6 Details of pellets of fractions enriched in plasma membrane. PACP staining. Fig. 3. Incubation with Con A-ferritin at pH 8. Clusters of ferritin particles (arrow) located between the vesicles (capping and agglutination). × 130 000. Fig. 4. Incubation with Con A-ferritin at pH 8 in the presence of Con A inhibitor: aMM. x 130 000. Fig. 5. Incubation with Con A-ferritin after treatment of membranes at pH 5. Note both profiles and glancing view (*) of the membrane labelled with ferritin particles, x 130 000. Fig. 6. Incubation with Con A-ferritin after treatment of membranes with 0.02% Triton X-100. Detail of the prof'fle of a membrane with an asymmetric distribution (on the outer face) of the ferritin particles, x 210 000.

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245 in the presence of 0.1 M aMM, membranes were free of ferritin (data n o t shown).

Effect of Triton X-I O0 on Con A-ferritin labelling o f PM and ER vesicles It has been shown that incubation of rat liver microsomes with low detergent concentrations leads to the selective release of proteins which represent the luminal content of the vesicles [18l. Since some of the released proteins have molecular weights higher than that of Con A (108 000 at pH 7.4), it was reasonable to expect that the same treatment applied to our membrane preparations would render them permeable to this lectin and perhaps to Man-Fer. Con A-binding sites on the luminal face of vesicles, if existent, could then be detected. This strategy has been applied with success in the case of glycoconjugates present in the luminal side of ER vesicles originating from rat liver [19]. In the present study, PM and ER vesicles from maize coleoptiles were treated with 0.02% Triton X-100 before to be tested for Con A binding. The ability of this procedure to readily change the permeability of the vesicles was checked by measuring the latency of UDPase, an enzyme which has been shown to be associated with both membrane preparations [ 15]. When PM vesicles are preincubated with Triton X-100 before Con A labelling, ferritin particles are seen to be exclusively distributed along the outer face of vesicles {Fig. 6). This detergent treatment leads to a complete loss of UDPase latency {see Ref. 12), indicating thus a change in membrane permeability. Figure 9 shows the effect of the same Triton concentration on the capacity of ER vesicles to bind Con A. Such a treatment results in an important modification of the morphological aspect of the preparation. Very long membrane strips with many ferritin particles dominate the pictures. Surfaces of membranes appear closely contiguous over considerable distances. Some smooth-surface vesicles are also seen. The increase in Con A binding caused by the detergent treatment parallels a change in membrane permeability as attested b y the loss of UDPase latency (see Ref. 12). Effect o f acid p H on Con A-ferritin labelling o f PM and ER vesicles In order to confirm the above results, it was interesting to test another

Figs. 7--9. Section of pellets of fractions enriched in endoplasmic reticulum. Uranyl acetate-lead citrate staining. Fig. 7. Incubation with Con A ferritin at pH 8. Presence of membranes single or join side by side. x 90 000. Fig. 8. Incubation with Con A-ferritin after treatment of membranes at pH 5. x 130 000. Fig. 9. Incubation with Con A-ferritin after treatment o f membranes with 0.04% Triton X-100. The large sheets o f membranes retain heavily the ferritin particles (arrows). x 9 0 000.

246 procedure to make the vesicles leaky, i.e. permeable to Con A and Man-Fer, consisting of an incubation at acid pH [11]. Both membrane preparations were therefore treated at pH 5.0 for 30 rain before the Con A-ferritin labelling procedure. As above, UDPase latency was measured and used as an index of membrane integrity for PM and ER vesicles. Figure 5 shows the effect of acid pH on Con A binding by PM vesicles. Ferritin particles are seen to be exclusively associated with the external face of the vesicles. The same treatment applied to ER vesicles induces a high increase in reactivity of membranes towards Con A-ferritin (Fig. 8). In contrast to the detergent treatment, the incubation at pH 5.0 allows retention of the vesicular form of membranes. In both cases [ 12], the acid treatment of preparations involves a complete loss of UDPase latency. D~CU~ION The two-step labelling method with Con A and IVian-Fer used in this report reveals evidence for Con A binding sites on the outer surface of P M vesicles isolated from etiolated maize coleoptiles, indicating thus the exist~ ence of available mannosyl and/or glucosyl residues. This histochemical technique is highly specific since no ferritingrains were present in control experiments carried out with aI~IM. It should be pointed out that P M vesicles labelled by Con A-ferritin were also reactive towards PACP. Ferritin particles were reproducibly found to be not randomly distributed over the surface of P M vesicles but associated into clusters.The aggregation of the vesicles which is observed in Figs. I--3, presumably results from the self,association of bound Con A molecules. Binding of Con A by P M vesicles from other plant species was recently reported elsewhere [20,21]. Pretreatment of P M vesicles by either low concentrations of Triton X-100 or low p H (procedures which make the vesicles leaky without solubihzation of membrane components [15,22] ) before subjection of the vesicles to Con A-ferritin binding did not alter the pattern of binding. Ferritin particles were stillseen to be exclusively associated with the outer side of the membrane of vesicles,with none found on the inner one (Figs. 5 and 6). Within the limits of resolution of the EM,~ytochemical method procedures used here, the results indicate that the glycoconjugates of the plant PM are asymmetrically distributed and located along the extracytoplasmic face of that membrane as in animal cells [1]. They also suggest that the PM-rich preparations from maize coleoptiles contain only right side-out vesicles. In contrast to PM vesicles, the ER-rich fraction bound small amounts of Con A, suggesting that few binding sites for this lectin are present on the outer face of the vesicles.As already discussed in the preceding paper [12], these available Con A binding sites could be associated with other membrane fragments which contaminate E R vesicles, especially Golgi membranes and/or tonoplast. Treatment of E R vesicles with 0.02--0.03% Triton X-100 before Con A-

247 ferritin labelling induced both a dramatic increase in Con A binding and a complete change in the morphology of the membranes. The vesicular shape of membranes disappeared to give very long strips closely contiguous over considerable distances and heavily labelled. To our knowledge, such morphological changes of membranes following a detergent t r e a t m e n t have n o t been reported elsewhere. We suggest t h e y result from some fusion phenomena. It should be pointed out that the treatment of rat liver microsomes with 0.04% sodium deoxycholate, an ionic detergent, does n o t alter the vesicular shape of membranes [19]. The same result was obtained after incubation of ER vesicles from maize at acid pH. In this latter case, a very high increase in the label of the membranes with Con A-ferritin was also observed. Thus, the most part o f Con A binding sites in the ER-rich fraction are not freely accessible to Con A and blan-Fer; they become available only after treatment of the vesicles by procedures which alter the membrane permeability, but w i t h o u t solubilizing the membrane components [ 10,11]. Within the limits of resolution of the EM-cytochemical m e t h o d procedures used here, the data suggest that most Con A receptors are located in membrane glycoconjugates which are exposed on the luminal side of ER vesicles. The conclusions obtained here are supported by our quantitative biochemical data found elsewhere [ 12 ]. ACKNOWLEDGEMENTS

This work was supported by the D ~Idgation G~n~mle A la Recherche Scientifiqueet Technique, grant No. 797 0783. REFERENCES 1 G.L. Nicolson and S.J. Singer, J. Cell Biol., 60 (1974) 236.

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