Concanavalin a binding as a tool in determining the sidedness of membrane vesicles from maize coleoptiles

Plant Science Letters, 30 (1983) 227--238

227

Elsevier Scientific Publkhers Ireland Ltd.

C O N C A N A V A L I N A BINDING AS A TOOL IN D E T E R M I N I N G THE SIDEDNESS O F MEMBRANE VESICLES FROM MAIZE COLEOPTILES M.A. H A R T M A N N , A. E H R H A R D T

and P. BENVENISTE

E.R.A. au C.N.R.S. No. 4~7 (Structure et fonction des membranes p@ricellulaires, Contr61e de la biosynth~}se des st~rols), Institut de Botanique, 28, rue Goethe, 67083-

Strasbourg Cddex (France)

(Received August 12th, 1982) (Revision received October 1st, 1982)

(Accepted October 26th, 1982)

SUMMARY

Plasma membrane(PM)- and endoplasmic reticulum(ER)-rich fractions from etiolated maize coleoptiles were assayed for their ability to bind concanavalin A (Con A). Evidence is presented for lectin binding sites in b o t h fractions, proving the presence of available mannosyl and/or glucosyl residues on the membrane surface of vesicles. Results from quantitative binding studies using [3H] Con A showed that Con A binding is specific, of high affinity and saturable. Compared to ER vesicles, PM vesicles b o u n d three times as much Con A. Con A binding by both fractions was also assessed after disruption o f the vesicle membrane by Triton X-100 or low pH. While a dramatic increase in binding capacity of Con A to ER vesicles was achieved after treatment with low concentrations of detergent or at pH 5.0, the level of Con A binding to PM vesicles was not altered, indicating that these treatments did n o t expose additional lectin binding sites. Results are discussed in relation to the sidedness of PM and ER vesicles. Plasma membrane -- Endoplasmic reticulum -- Concanavalin A binding -- Sidedness of membrane vesicles

Key words

INTRODUCTION F e w enzymes have been shown to be associated with the P M of plant cells. A m o n g them, our attention has been focused on the UDP-glucose

sterol-0-D-glucosyltransferase and the uridine 5'~iiphosphatase (UDPase) Abbreviations: ER, endoplasmic reticulum; PM, plasma membrane; Con A, concanavalin A;aMM, a-methyl-D-mannoeide; UDPase, uridine 5'-diphosphatase. 0304-4211/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.

Printed and Published in Ireland

228 whose activities have been detected in PM-rich preparations from etiolated maize coleoptiles [1--3]. A prerequisite for assigning a physiological role to both enzymes is to determine the orientation of their active sites in the transverse plane of PM. Such a purpose is difficult to achieve because of the absence of surface-specific enzymic markers for monitoring the integrity and orientation of vesicles arising from grinding of plant tissues. When subjected to homogenization, the PM is extensively disrupted and gives a mixture of fragments having possibly different orientations i.e.: (i) closed vesicles oriented with the cytoplasmic face turned towards the interior of vesicles (right side-out vesicles); (ii) closed vesicles with an opposite orientation (inside-out vesicles); (iii) unsealed vesicles and/or sheets. To evaluate the sidedness of vesicles originating from the PM after fragmentation of maize coleoptile cells, we decided to use Con A as a probe for the investigation of carbohydrate chains. These latter can be considered as orientation markers since, in animal cells, membrane glycoconjugates (i.e. glycoproteins and glycolipids) have been shown to be asymmetrically distributed in the transverse plane of membranes, the carbohydrate residues being located exclusively on the extracellu!ar face of the PM [4,5]. Con A is specific for a-D-mannopyranosyl and a-D-glucopyranosyl residues [6], which are monosaccharides well represented in biological membranes, although little is known about the carbohydrate composition of plant PM. However, an indirect proof of the presence of these carbohydrate residues on the surface of plant cells is given by the ability of plant protoplasts to blnd Con A [7--10]. We have recently succeeded in isolating two membrane fractions from etiolated maize coleoptiles, the first one enriched in PM and the second one in ER [1,2]. In the present work, both fractions were assayed for their ability to bind [3HI Con A. Evidence is presented for the presence of Con A receptor sites in PM- and ER-rich fractions. The capacity for Con A binding by both fractions was also examined after disruption of membrane integrity in order to detect some binding sites not freely accessible to the lectin. Some preliminary aspects of this work have appeared elsewhere [11]. After this work was completed, we became aware of a report from Berkowitz and Travis [12] on Con A binding by isolated PM from soybean roots. 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 fr~ctions were isolated as described previously [1]. Both membrane fractions were washed free of sucrose by repeated centrifugation and then restmpended in 0.1 M Trb~HCI (pH 8.0) containing 1 mM mercaptoethanol.

229

Binding of Con A to membrane fractions Binding experiments were carried o u t in duplicate by incubating membrane fractions (50--100 ~g of membrane protein) with the desired a m o u n t of Con A in 0.2 M NaCI in a final volume of 0.5 ml. [3H] Con A (48 Ci/ mmol, New England Nuclear) was diluted with unlabelled Con A just before use. A b o u t 25 000 cpm of tritiated Con A were introduced per assay. The Con A concentrations used in assays are indicated in the legends of figures. Samples were incubated at 25°C for 1 h, then centrifuged at 35 000 × g for 30 min in a R 25 Beckman rotor. Pellets were stored at 4°C overnight in 0.3 ml of 4% (w/v} Triton X-100 in 0.1 M Tris--HC1 (pH 8.0). The suspension and a 0.2 ml Triton solution wash of the tubes were transferred to counting vials. After addition of 10 ml Bray scintillation fluid, the radioactivity was measured. Control experiments were performed with 50 mM aMM in addition to the above constituents. Specific Con A binding was evaluated as the difference between the amounts of radioactivity b o u n d to membranes in the presence and absence of aMM. Effect of Triton X-I O0 and low pH on Con A binding by PM and ER vesicles Membrane fractions were treated with low concentrations of Triton X-100 (0--0.05%, w/v) for 15 min at 30°C or at different pH-values (8.0, 6.5, 5.5 and 4.0) for 30 rain at room temperature. After centrifugation of the incubates at 100 000 × g for 30 min, pellets were resuspended in 0.1 M Tris--HC1 (pH 8.0) and tested both for Con A binding and UDPase activity. UDPase assays They were performed as indicated in Ref. 3. UDPase activity was measured in the absence and presence of 0.02% or 0.04% Triton X-100 for PM and ER vesicles, respectively. Protein determinations Proteins were determined by the m e t h o d of L o w r y et al. [13]. RESULTS

General features o f Con A binding by PM and ER vesicles F r o m preliminary experiments, suitable conditions for Con A binding by PM and ER vesicles from maize coleoptiles were shown to be the following: pH 7.5, incubation time: 1 h, temperature: 25°C, absence of Mg 2÷, Ca 2+ and Mn 2+ ions (see Discussion). To evaluate the effect of lectin concentration on Con A binding, an isotopic dilution method was used. This consisted to introduce in all the assays a same fixed a m o u n t of [3H] Con A (25 000 cpm, 8.9 • 10 -9 M) with increasing concentrations of unlabelled Con A (10 - 8 10 -4 M) to a constant protein concentration. Dilutions were performed just prior to assay. After incubation, bound and free Con A were separated by centrifugation as described in Materials and Methods. As shown in Fig. 1, PM vesicles bind [3H]Con A very effectively. The total binding of [3H]Con A

230 total binding 41

o M

E

O. v

10 ~

•

U

Z 5

...._, J

-e

i

-7

I

-6

i

-s Iog[¢o

=

*l

Fig. 1. Saturation curve of Con A binding to PM vesicles. In all the assays, a same fixed amount of [ s H ] C o n A (25.000 cpm, 8.9 • 10-' M) and various amounts of unlabelled Con A (10"~--10 -4 M) were incubated in the l~esence of a constant PM protein concentration (50 , g ) . See detaik in MateriaLs and Methods.

decreases with increasing concentration of unlabelled Con A, i.e.tritiated Con A molecules compete with native Con A molecules for specific binding sites.A complete saturation of these latterby cold Con A is observed for Con A concentrations higher than 10 -4 M. At this concentration, the residual radioactivity which is measured represents the non-specific binding of Con A to P M vesicles. Alternatively, this can be determined by adding a M M to assays. The values obtained for non-specific Con A binding were nearly the same in both procedures and constituted less than 1 0 % of the total radioactivity bound to membranes. The binding of [3H] Con A by P M vesicles increased linearly with respect to the membrane protein concentration up no more than 9.5% of the total amount of lectin introduced into the incubation mixture was bound to membranes (data not shown). If the data described in Fig. I are plotted according to the method of Scatchard ~14], a linear relationship is reproducibly obtained but only over a range of Con A concentrations from 2 • 10 -7 to 10 -4 M (Fig. 9.A). Under these conditions, using a molecular weight of 110 000 for Con A, the maximal amount of lectin bound per m g of P M

231

A

PM

+

21 ~

~

~

k

~

.

.

.

~

O ~

*

I'-'~ *~~t...._.

~

.

.

.

.

--

Triton X-tO0 Triton X-100

~ ,

.

s

ER

~ ~3 ,.a

2

5

tO

15

20

25

30

ConA bound Mxl0* Fig. 2. Sc~tchard plots of specific [~H]Con A-binding to PM and ER vesicles, in the a b s e n c e a n d p r e s e n c e o f T r i t o n X - 1 0 0 (0.02% a n d 0,03% for PM a n d E R vesicles, respectively).

protein is shown to be around 1 0 -9 mol. Thus the Con A binding by P M vesicles is a saturable process and of high affinity (Kd = 1.2 ~ M ) (Table I). Similar experiments were performed with an ER-rich fraction from maize coleoptiles. The results showed that significantCon A binding was achieved. A saturation curve similar to that obtained with P M vesicles was exhibited TABLE I S P E C I F I C B I N D I N G O F [ 3 H ] C O N A TO ER- A N D PM-RICH F R A C T I O N S F R O M MAIZE COLEOPTILES Q u a n t i t a t i v e d a t a f r o m S c a t c h a r d plots. PM

ER

- T r i t o n X-100

K d = 12 • 10 "7 M L M = 970 p m o l / m g p r o t . a

K d --- 8.5 • 10"7M L M = 320 p m o l / m g p r o t .

+ Triton X-100 b

Kd = 12" 10-"M L M = 970 p m o l / m g p r o t .

K d --- 8.5 • 1 0 ~ M L M = 1130 p m o l / m g p r o t .

aMaximal a m o u n t o f lectin b o u n d per m g o f PM p r o t e i n . b 0 . 0 1 - - 0 . 0 3 % , w/v.

232

(data not shown), proving that Con A binding by E R vesicles is also a saturable process and of high affinity (Kd ffi0.85 ~M). Binding was shown to be inhibited by 50 m M a M M , indicating that the interaction between E R vesicles and the lectin is specific for Con A. From Scatchard plot data, it was calculated that there are about 0.32 × 10 -9 mol of Con A bound per m g of protein (Fig. 2B). Compared to P M vesicles, the ER-rich fraction binds less than three times as m u c h Con A (Table I). Effect o f Triton X-IO0 on Con A binding by PM and E R vesicles Since Con A is not expected to cross membranes, only those carbohydrate residues located on the extracytoplasmic face of right side-out vesicles of PM are susceptible to interaction with the lectin. If some PM vesicles have not retained their in vivo orientation, then some carbohydrate residues not freely accessible to Con A must exist, unless membrane integrity is disrupted. In order to detect such binding sites for Con A in both membrane fractions, PM and ER vesicles were treated with different low concentrations of Triton X-100 (up to 0.05%, w/v) prior to assaying for Con A binding. To test the ability of the detergent to readily make the vesicles leaky, the permeability characteristics of vesicles have been investigated by measurement of the latency of UDPase. In a previous work, evidence was indeed presented for a latent UDPase in both PM and ER vesicles from maize coleoptiles. The per cent of latency expressed as [1 - (activity in intact vesicles/activity in disrupted vesicles)] X 100 was shown to be 40--50% and 85% in PM- and ER-rich fractions, respectively [3]. Table II indicates that whatever Triton concentration has been used in the pretreatment, no increase in specific binding of Con A by PM vesicles was achieved. Under the same assay conditions, the patent activity of T A B L E II E F F E C T O F A P R E T R E A T M E N T O F PM WITH T R I T O N X-100 ON C O N A - B I N D I N G A N D UDPs~e A C T I V I T Y % T r i t o n X-100 in the p r e t r e a t m e n t

Con A specific binding a UDPase a c t i v i t y b - T r i t o n X-100 + T r i t o n X-100

% Latency c

0

0.005

0.01

0.02

0.03

31 500

31 000

30 000

24 000

21 000

1 1.6

1.6 2.0

1.6 1.6

0.5 0.8 40

1 1.6 40

~

40

20

0

*Specific binding of C o n A : c p m / m g o f protein. b U D P u e activities: n m o l / s / m ~ o f protein. cpercentage of latency: [ 1 - (activity in intact vesicles/activity in disrupted vesicles)] x 100.

233 TABLE III E F F E C T OF A P R E T R E A T M E N T OF ER WITH TRITON X-100 ON CON A-BINDING AND UDPase ACTIVITY % Triton in the pretreatment 0

0.01

0.02

0.03

0.04

0.05

Con A specific binding a

9700

15 000

14 500

17 300

18 300

25 000

UDPase activity b - T r i t o n X-100 + Tr i t o n X-100

0.5 4

0.7 3.8

1 3.5

%L~ency c

90

80

70

2 3 35

2.6 3 15

3.5 2.6 0

aSpecific binding of Con A: cpm/mg of protein. bUDPase activities: nmol/s/mg of protein. Cpercentage of latency: [1 - (activity in intact vesicles/activity in disrupted vesicles)] × I00.

UDPase, i.e. the activity which was measured in the absence of detergent, progressively increased and was accompanied by a decreasing per cent of latency. No latency was observed after a treatment of PM vesicles with 0.03% Triton X-100, providing evidence that the permeability barrier of the vesicles has been disrupted to allow the substrate access to active sites located on the internal surface of vesicles. In the PM-rich fraction, all the Con A binding sites are therefore accessible to the lecti~, with vesicles rendered permeable or not to UDP. In contrast to PM vesicles, treatment of E R vesicles with Triton resulted in a dramatic increase in specific Con A binding. In Table III, it can be seen that the pretreatment of membranes with 0.05% Triton X-100 involves a 2.5--3-fold stimulation of Con A binding and also a complete loss of UDPase latency, suggesting that some Con A binding sites are located on the luminal face o f vesicles as the most part of UDPase active sites [3]. However it was important to be certain that the increasing amounts of Triton X-100 did n o t influence the kinetic parameters of Con A binding to PM and ER vesicles. Consequently, specific binding of [SH] Con A was measured in the absence and presence of 0.01% and 0.03% Triton for PM and ER vesicles, respectively, as a function of Con A concentration to determine the affinity of binding sites for Con A and the number o f these sites. As indicated by Scatchard plots (Fig. 2 and Table I), Kdvalues appear to be unaffected by the detergent. Thus, the increase in Con A binding after treatment of ER vesicles with Triton X-100 results effectively from a change in the total n u m b e r of binding sites and n o t from a change in their affinity for the lectin. These additional binding sites become accessible to Con A only after disruption of the vesicle membrane by the detergent.

234

TABLE IV EFFECT OF A PRETREATMENT OF PM WITH ACID pH ON CON A-BINDING AND UDPase ACTIVITY pH of the pretreatment

Con A specific bindings

8

6.5

5.5

4

40 000

47 000

48 000

53 000

UDPase a c t i v i t y b

- T r i t o n X-100 +Triton X-100 % Latency c

1 1.7 40

1.4 1.8 20

1.4 1.8 20

1.3 1.3 0

aSpecific binding of Con A: cpm/mg of protein. bUDPase activities: nmol/a/mg of protein. Cpercentage of latency: [1 - (activity in intact vesicles/activity in disrupted vesicles)] x 100.

Effect o f low pH on Con A binding by PM and ER vesicles Similar experiments were performed using an alternative procedure of membrane disruption in attempts to extend and verify the results in preceding sections. The treatment of microsomes with low pH has been indeed shown to result in a loss of latency for enzymatic activities associated with the cistemal face of vesicles [15]. Moreover, an advantage in such a proceTABLE V EFFECT OF A PRETREATMENT OF ER WITH ACID pH ON CON A-BINDING AND UDPase ACTIVITY pH of the pretreatment

Con A specific bindings

8

6.5

5.5

4

35 000

61 000

86 000

95 600

UDPase a c t i v i t y b

- T r i t o n X-100 +Triton X-100 % Latency c

0.7 3.4 80

1.3 3.2 60

2.2 3.3 35

1.0 1.0 0

aSpecific binding of Con A: cpm/mg of protein. bUDPese activities: nmolls/mg of protein. CPercentage of latency: [1 - (activity in intact vesicles/activity in disrupted vesicles)] x 100.

235

dure is not to involve the solubilization of membrane constituents. Thus, both membrane fractions were incubated at different pH-values (8.0, 6.5, 5.0 and 4.0) before measuring Con A binding. As previously, latency of UDPase was used as an index of membrane integrity for P M and E R vesicles.Treatment of P M vesicles with acid p H was shown to slightly increase the extent of Con A binding and also to induce a progressive increase in membrane permeability toward U D P (Table IV). A complete loss of UDPase latency was observed after a preincubation of P M vesicles at p H 4.0. In contrast to P M vesicles, Con A binding by E R vesicles was demonstrated to be strongly dependent on the p H of the incubation mixture. When treated at p H 4.0, E R membranes bound 2.5--3-fold more Con A than the control incubated at p H 8.0 (Table V). This treatment also allowed U D P to freely penetrate the vesicles as attested by the loss of UDPase latency. Scatchard analysis of Con A binding to p H 5.0-treated and control m e m branes indicated that the increase in binding after incubation at acid p H results from an increase in the absolute number of high affinity binding sites and not from a change in their affinity (data not shown). DISCUSSION

In the present work, evidence is presented for Con A binding by P M and E R vesicles isolated from maize coleoptiles, proving the existence of available mannosyl and glucosyl residues in both fractions. Optimal assay conditions were established by monitoring the effect of lectin concentration, incubation time and membrane protein concentration on the specific binding of [3H] Con A by membrane preparations. Assays were performed in the absence of divalent cations to avoid events of membrane fusion, which were found to be strongly dependent on M n 2÷ and Ca 2+, even up at concentrations less than 1 m M [16]. W h e n these cations are omitted from the incubation medium, specific binding of Con A is reduced by 20--30% with respect to a control carried out in the presence of i m M Ca 2÷ and M n 2÷ (data not shown). Results from quantitative binding studies clearly indicate that the interaction between Con A and membranes is a saturable process in respect to Con A (Fig. 1), of high affinity (Kd around 1 pM) and specificity (inhibited by aMM). Of particular interest in the present study is the finding that [3H] Con A molecules are able to compete with unlabeUed Con A molecules for specific binding sites. A complete saturation of these latter by unlabeUed lectin is observed for Con A concentrations higher than 10 -4 M. Scatchard analysis of the binding data gave no straight lines, suggesting that the interaction of Con A with membranes from maize coleoptiles is quite complex and that multiple binding sites of differing affinity probably exist in both PM- a n d E R - r i c h f r a c t i o n s as in a n i m a l m e m b r a n e p r e p a r a t i o n s [ 1 7 ] . H o w ever, it s h o u l d be p o i n t e d o u t t h a t a linear r e l a t i o n s h i p was r e p r o d u c i b l y o b t a i n e d o v e r a r a n g e o f C o n A c o n c e n t r a t i o n s f r o m 2 • 10 -7 t o 10 -4 M

(Fig. 2).

236

The presence of Con A binding siteson the external surface of P M from intact plant protoplasts [7--10] or isolated vesicles [18,19] had been previously demonstrated. The results presented here, along with similar measurements by Berkowitz and Travb [12], provide some kinetic characterization of Con A binding to an isolated plant P M fraction. The m a x i m u m number of Con A binding sitesfound on P M vesiclesfrom maize coleoptiles, i.e.around 1 nmol/mg of protein, is near the values reported for P M from lymphocytes [20]. Since lectins neither cross the membrane nor destroy the permeability barrier of closed vesicles,only those carbohydrate residues exposed on the extracytoplasmic surface of right side-out vesiclesare susceptible to interact with Con A unless the permeability barrieris altered. The absence of an effect of Triton X-100 on the Con A binding capacity of isolated P M suggest that most of the carbohydrate residues of P M vesicles are accessible to Con A, i.e.externally disposed as in intact cells.The slight decrease in lectin binding by P M vesicles treated with Triton concentrations higher than 0.02% was probably due to a solubilization of some membrane or luminal glycoproteins [21]. Likewise, acid pH-treated P M vesicles did not exhibit important changes in specific binding of [3H] Con A. The increase of the number of Con A binding sites,which was observed at p H 4.0 (Table IV), is probably not significant and strongly differs from that obtained for E R vesiclesunder the same conditions (Table V). The nature of Con A receptor siteshas not been elucidated. It must be borne in mind that the assay employed is a functional one that measures only those molecules capable of active binding. The possibility that inert or non-functional binding sites may exist cannot be excluded. Since no additional lectin binding sites become accessible even after treatment of PM vesicles with detergent or low pH, it can be argued that all the vesicles of the PM-rich fraction from maize coleoptiles have the same right side-out orientation. According to this hypothesis, the enzymatic sites of UDPase, which are freely accessible to UDP, would be associated to leaky PM vesicles, indicating that UDPase would be an endoenzyme. The ability of intact ER vesicles to bind Con A may appear surprising since, in animal systems, it is known that rough ER and nuclear membranes bind only small amounts of Con A [22,23]. To explain this particular situation in our system, it is suggested that the available Con A binding sites in the ER-rich fraction are located on other kinds of membranes which contaminate this fraction such as plastid and amyloplastid envelopes, Golgi membranes and probably tonoplast fragments [1,2]. The orientation of the vesicles originating from these organelles is not known. It has recently been shown that plastid membranes either do not contain significant levels of glycoproteins [24] and do fail to react with Con A [25,26]. However, according to Schneider and Sievers [27], the surface of the starch grains would have some affinity for Con A. One possibility is that some Con A binding sites could be associated with inside-out vesicles of tonoplast [25]. Finally, the binding sites freely accessible to the lectin might also reflect

237 s o m e d i s r u p t i o n o f isolated E R vesicles. In a n y w a y , m o s t o f the g l y c o c o n jugates (66%) o f the ER-rich f r a c t i o n b e c o m e available t o Con A o n l y in the p r e s e n c e o f low c o n c e n t r a t i o n s o f T r i t o n X - 1 0 0 (Table III) or at acid pH (Table V). T h e o b s e r v a t i o n t h a t c r y p t i c lectin binding sites u n m a s k e d b y these t r e a t m e n t s e x h i b i t the same a f f i n i t y f o r [3H] Con A as t h o s e p r e s e n t in u n t r e a t e d vesicles (Fig. 2) suggests t h a t the increase in specific [3H] Con A 'binding caused b y d e t e r g e n t or low pH really results f r o m an increase in the a b s o l u t e n u m b e r o f lectin binding sites. As s h o w n in Tables III and V, the increase o f Con A binding closely follows the loss o f UDPase l a t e n c y , suggesting t h a t these a d d i t i o n a l c r y p t i c binding sites f o r Con A originate f r o m intravesicular sites. In this case, if we assume t h a t E R m e m b r a n e s retain t h e i r o r i e n t a t i o n f o l l o w i n g grinding o f coleoptiles, m o s t o f the carboh y d r a t e residues w o u l d have a luminal localization as in animal cells [ 2 3 ] . E l e c t r o n m i c r o s c o p y d a t a c o n c e r n i n g the t o p o l o g y o f t h e c a r b o h y d r a t e residues o f E R and PM vesicles will he r e p o r t e d in the n e x t p a p e r [ 2 8 ] . ACKNOWLEDGEMENTS This work was supported by the Ddldgation G~nSrale ~ la Recherche Scientifique et Technique grant No. 797 0783. REFERENCES 1 M.A. Hartmann-Bouillon and P. Benveniste,Phytochemistry, 17 (1978) 1037. 2 M.A. Hartmann-Bouillon, P. Benveniste and J.C. Roland, Biol.Cell.,35 (1979) 183. 3 M. M'Voula-Tsieri,M.A. Hartmann-BouUlon and P. Benveniste,Plant Sci.Lett.,20 (1981) 379. 4 G.L. Nicolson and S.J. Singer, J. Cell Biol., 60 (1974) 236. 5 I. Virtanen, A. Miettinen and J. Wartiovaara, J. Cell Sci., 29 (1978) 287. 6 I.J. Goldstein, C.M. Reichert and A. Misaki, Ann. N.Y. Acad. Sci., 234 (1974) 283. 7 J. Burgess and P.J. Linstead, Planta, 130 (1976) 73. 8 F.A. Williamson, L.C. Fowke, F.C. Constabel and O.L. Gamborg, Protoplasma, 89 (1976) 305. 9 J.C. Chin and K.J. Scott, Ann. Bot., 43 (1979) 33. 10 F.A. Wflliamson, Planta, 144 (1979) 209. 11 M.A. Hartmann-Bouillon, A. Ehrhardt and P. Benveniste, Plasmalemma and Tonoplast: Their Functions in the Plant Cell, Elsevier, Amsterdam, 1982, p. 163. 12 R.L. Berkowitz and R.L. Travis, Plant Physiol., 68 (1981) 1014. 13 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. 14 G. Scatchard, Ann. N.Y. Acad. Sei., 51 (1949) 660. 15 J.A. Hanover and W.J. Lennarz, J. Biol. Chem., 255 (1980) 3600. 16 E.A.H. Baydoun and D.H. Northcote, J. Cell Sci., 45 (1980) 169. 17 P. Cuatrecasas, Biochemistry, 12 (1973) 1312. 18 W.F. Boss and A.W. Ruesink, Plant Physiol., 64 (1979) 1005. 19 R.L. Travis and R.L. Berkowitz, Plant Physiol., 65 (1980) 871. 20 K. Resch, A. Loracher, B. Mahler, M. Stoeck and H.N. Rode, Biochim. Biophys. Aeta, 511 (1978) 176. 21 G. Kreibieh and D.D. Sabatini, J. Cell Biol., 61 (1974) 789.

238 22 23 24 25 26 27 28

T.W. Keem~-, W.W. Franke and J. Kartenbeck, FEB8 Lett., 44 (1974) 274. E. Rodriguez-Boulan, G. Kreibich and D.D. Sabatini, J. Cell Biol., 78 (1978) 874. K. Keegstra and K. Cline, Plant Physiol., 70 (1982) 232. S.E. Frederick, B. Nies and P.J. Gruber, Planta, 152 (1981) 145. C.R. Stocking and V.R. Franceschi, Am. J. Bot., 68 (1981) 1008. E.M. Schneider and A. Sievers, Pianta, 152 (1981) 177. M.A. Hartmann, P. Benveniste and J.C. Roland, Plant Sci. Lett., 30 (1983) 239.