Maltose excretion by the symbiotic CA/ore//a of the heliozoan fur/acea

P l a n t a ( 1 9 9 0 ) 181:593-598

Planta

( Springer-Vcrlag 1990

Maltose excretion by the symbiotic CA/ore//a of the heliozoan fur/acea

Bettina Matzke, EMsabeth Schwarzmeier, and Eckhard Loos^

L e h r s t u h l fur ZeHbiotogie und Pflanzcnphysiotogic, Univcrsitat Regensburg, Univcrsitatsstrasse 31, D-8400 Regensburg.

Federal R e p u b l i c o f G e r m a n y

Abstract. C/;/or(?//a sp. strain 3.83, a symbiotic (Vy/fw/Za isolated from the heliozoan

ex- creted between 8% and 16% of assimilated ^ C 0 2 as maltose in the light (15000 lx), with a p H optimum around 4.8. This percentage increased when the illumin- ance was lowered (36% at 1 700 lx). Release of [*^C]mal- tose continued in darkness and could be inhibited by the uncoupler carbonyl cyanide /7-trifluoro-methoxy- phenylhydrazone and by diethylstilbestrol. Net efflux of maltose was observed even at a concentration ratio of extracellular/intracellular maltose of 7.8. Exogenous [*^C]maltose (5 m M ) was taken up by the cells with a rate < 2 % of that of simultaneous maltose release, indicating a practically unidirectional transport. It is concluded that maltose excretion is an active-transport process.

Key words: C/yAw/Za (symbiosis) - Maltose excretion (CA/ar^/A?, symbiosis) - Symbiosis (C/?/^<?//%-/Ira/?//?<?- q y ^ ) - Transport, active

Introduction

Maltose excretion has been shown to occur in the sym- biotic Chlorellae from green T/y&a and from the ciliates

P<2n377^c;*M??2

and 57<?/i;or (see Reisser and Wiessner 1984). The influence of some physicochemical factors on this process has been investigated (Muscatine 1965;

Cernichiari e t a l . 1969) and the origin of excreted mal- tose has been studied (Cernichiari et al. 1969; Zieseniss et al. 1981). Scanty data, however, exist on the possible active nature of the excretion process (Zieseniss 1982).

It is the purpose of this work to present briefly the growth characteristics of a CAAw/Za isolated from the Heliozoan ^ c< 3 M ? / ? o c y a n d to characterize the maltose

* To w h o m correspondence should be addressed

^M>r^/a7;o/M.- D E S = d i e t h y l s t i l b e s t r o l ; F C C P = carbony! cyanide /7-trinuoromethoxypheny! h y d r a z o n e ; p . c —packed cells

excretion by this alga, also with respect to an active transport of this sugar.

Materia! and methods

(7;/^rr/A/ sp. strain 3.83. the endosymbiont from

/ ^ ^ Y 7 / 7 / / ? r ^ i . \ / / . \ / M r / ^ ^ v , was obtained from S a m m l u n g fur A l g e n - kulturcn G o t t i n g e n . F R O . The alga was grown under axenic c o n d i - tions in i i q u i d m e d i u m c o n t a i n i n g the foHowing salts in m o ! 1 ^ ( N H J ^ S O i , 5-10 \ K H 2 P O 4 - 5 10 ' ; M g S O ^ . 10 ' ; C a C ^ , 10 ^. F c S O ^ and trace elements were as given by K u h ! (1962). F o r l i q u i d starter cultures the growth m e d i u m was enriched with 0 . 5 % meat extract ( M e r c k . D a r m s t a d t . F R C ) . Other c o n d i - tions o f growth have been described previously (Fischer et al.

1989). M a i n t e n a n c e cultures were kept in petri dishes on culture m e d i u m with 0 . 5 % meat extract and 1% agar; they were stored close to a w i n d o w and shaded from direct sunlight. The ccHs were harvested after 3 6 d at cell densities between 1.0 and 2.0 ul packed cells (p.c.)-ml ' and were resuspended in 50 m M citric acid tri- s o d i u m citrate buffer p H 4.8 at a density o f 2 u! p . c m ! * un!css indicated otherwise.

/ / K ' M/ 7 < 7 f / f J f 7 f ? / ( 7! / ( W / / ; V 117//? ' ^ ( d i , ('.\7//?7r/f/f'/7 / / 7 ( ( V/ 7 f; ; ' ( V / / f ' / 7 a n J (\Y(7T//f)/? rj/ ^;7(7/i.\7.s A / / ? r/ f r/ r . \ r n^ / ^ / 7

H f / . s . T h e procedure has been described in detail e!sewhere (Fischer

et a l . 1989). Briefly, 1 ul o f packed ce!!s suspended in 0.5 ml 50 m M citric acid trisodium citrate buffer ( p H 4.8) were shaken (260 rpm) at 28° C in an atmosphere containing 0.9% O2 (v v) with a specific radioactivity o f 7.4 k B q umo! The illuminance was 15000 lx and the incubation time was 1 h. un!css indicated otherwise. R a d i o a c t i v i t y o f cells was determined by scintillation c o u n t i n g o f a cell hydrolysate and ['**C]ma!tosc excretion by T L C o f the desalted medium, m a k i n g use o f a TL(L-scanning device ( L B 284; B c r t h o l d , W i l d b a d . F R G ) .

D( V ( V / 7 7 f / 7; / f/ ^ A l ^ f ' A / 7 Y V f f / / ! ^ V r /77f///fAS<' fV/7<V f j / //7fl7/((V/7v/(V/* / 7 7 ^ / / f A \ f . . y M r f y w ^ / ! v r ^ A f / ^ / 7 V ( Y A^ - 6- / ? / 7 ^ . \ / ? / 7 ^ / f . M a l t o s e in the m e d i u m was determined after cleavage with x-g!ucosidasc using the h e x o k i - nasc/g!ucose-6-phosphatc dehydrogenase system ( G u t m a n n 1974).

F o r assays o f intracellular sugars, aqueous cell extracts were ob- tained a c c o r d i n g to the method o f Bicleski (1982) as modified by Fischer et a!. (1989). The yield o f extraction was checked by a d d i n g a k n o w n amount o f carrier-free [*^C]maltosc to the initial extrac- tion mixture and by c o u n t i n g an aliquot at the end o f the proce- d u r e ; 8 3 % o f the radioactivity was recovered. T h e content o f glu- cose+glucosc-6-phosphatc o f the aqueous extracts was determined

enzymatically w i t h hexokinase/glucose-6-phosphate dehydrogenase (Bergmeyer e t a l . 1974). T h e sucrose content was obtained after i n c u b a t i o n w i t h invertase o n the basis o f the same method (Berg- meyer and Bernt 1974) and the maltose content after a n a d d i t i o n a l i n c u b a t i o n w i t h a-glucosidase ( G u t m a n n 1974). T h e cell v o l u m e was determined as the volume inaccessible to soluble starch (Fischer et a l . 1989); this p o l y m e r showed no measurable degrada- tion o r uptake by the cells within the time span o f its a p p l i c a t i o n .

57/;'<ro7?^-o;7 <r^Mfn/M^%/;'o/? #n<3f /i7?ra?;'oH / b r < ^ / / F o r sili- cone-oil centrifugation, 3 m l o f 1 5 % (w/v) trichloroacetic acid, 3 m l silicone o i l (type A R 200; F l u k a , Buchs, Switzerland) a n d 1 m l o f 50 m M citric a c i d - t r i s o d i u m citrate buffer p H 4.8 were pipetted i n the given order i n t o a siliconized centrifuge tube a n d precentri- fuged for a short time to o b t a i n sharp phase boundaries. T h e algal suspension (250 u l p . c ) , i n 5 m l o f 50 m M citric a c i d - t r i s o d i u m citrate buffer p H 4.8, was added to the top o f the tube. After 5 m i n centrifugation at 1 2 0 0 - g the upper a n d m i d d l e phases were removed, the sedimented cells were resuspended i n the lower phase and were kept at 4 ° C for 30 m i n . After another centrifugation the supernatant ( — cell extract) was taken and repeatedly extracted with diethyl ether to remove trichloroacetic acid. After concentra- tion to an appropriate volume a n d adjustment o f p H , maltose was determined as described above. In a parallel experiment, the same a m o u n t o f cells was vaccum-filtered o n a cellulose-nitrate membrane filter (0.45 um) a n d washed three times w i t h ice-cold 50 m M citric a c i d - t r i s o d i u m citrate buffer p H 4.8. T h e filter w i t h the cells was extracted for 30 m i n at 4 ° C i n 3 m l o f 1 5 % (w/v) trichloracetic a c i d ; the f o l l o w i n g procedures were as above.

ResuMs

AforpAo/ogy

grotW/z

^p.

The cells contained a cup-like chloroplast and were egg-shaped or almost spherical in young and mature stages, respectively; the diameter of mature cells was approx. 7.5 urn. Normally, four autospores were released

0 !M,MT M S G

T V V T V

''.-.-'-it-vr

0 !M.MT M S G V V V T V

E a.

o E D

Time [h]

Fig. 1. K i n e t i c s o f ^ C - l a b e l i n g o f cells a n d o f excretion products o f symbiotic C/i/prp/Za sp. 3.83. A A , radioactivity i n cells; *

* , r a d i o a c t i v i t y in the m e d i u m ; o O, radioactivity o f uncharged material in the m e d i u m . T h e rate o f *^C02 fixation was 541 u m o l - h " * - ( m l p . c . ) " * . After the light p e r i o d , one sample was centri- fuged, the cells were resuspended i n fresh buffer, transferred into a darkened v i a l w i t h a i r as gas phase a n d were shaken. E v e r y half h o u r the m e d i u m was removed for c o u n t i n g and was replaced by fresh buffer; the c u m u l a t e d counts were plotted i n the g r a p h

Fig. 2a, b. Scans for radioactivity o f thin-layer chromatograms.

a E x c r e t i o n products o f C/z/oyr//^ sp. 3.83 at p H 4.8; the m e d i u m was treated w i t h m i x e d ion-exchange resins before separation (Fischer e t a l . 1989). b A n a l y s i s o f the peak at the p o s i t i o n o f maltose i n a. T h e peak material was eluted a n d incubated for 2 h at 37° C i n a total v o l u m e o f 0.5 m l 50 m M N a - c i t r a t e buffer p H 4.8 c o n t a i n i n g 7 U amyloglucosidase (Boehringer, M a n n h e i m , F R G ) , treated w i t h a mixed-bed i o n exchanger ( M B - 3 ; Serva, H e i - delberg, F R G ) a n d was rechromatographed. In a and b, silica-gel- coated a l u m i n u m sheets were used w i t h acetone: n - b u t a n o l : H 2 O = 7 0 : 1 5 : 1 5 (by vol.) as solvent system. T h e positions o f reference substances are i n d i c a t e d : (7 —glucose, 5*= sucrose, A f = maltose, j M T = m a l t o t r i o s e ; / M = isomaltose; 0 = o r i g i n

from mother cells. G r o w t h was exponential up to a cell density o f approx. 7.7-10^cells-ml"* with a doubling time of 14 h. The addition of the combined vitamins B i (1 m g - m l ' * ) and B12 (0.1 mg-1"*), which is essential for other symbiotic Chlorellae (ReiBer 1975; ZieseniB et al. 1981; M u c k e 1985), did not influence the growth rate of C/i/or^Z/a sp. 3.83.

F;'x<3?;'6)f2 6?/ ^C6^2 ?' ^-xc?T//(3f3 a^a? <2??a/y

/ y r o ^ M C ' ^ .

Assimilation of ^ C 0 2 was linear for at least

1 h and was accompanied by excretion of radioactive material (Fig. 1). This amounted to between 10 and 20%

of total fixed ^ C in several experiments. U p o n darken- ing, excretion continued with little change in rate and, after 2 h in darkness, had reached a value of >30%.

After treatment with mixed-bed ion exchangers, most of the labeled excretion products (typically 80% of them) were still in solution and, therefore, are considered to represent uncharged compounds. Thin-layer chromatog- raphy of these showed a conspicous peak at the position of maltose (Fig. 2a). After elution, treatment with amy- loglucosidase and rechromatography the position of ra- dioactivity had changed to that of glucose (Fig. 2 b).

Thus, maltose is obviously the main excretion product of the

endosymbiont.

f<37*<3?n^7*3

M?/7M ^f?(r;'/?g

The depen- dence of maltose excretion and ^ C 0 2 fixation on the p H of the medium is shown in F i g . 3. Maximum rates of maltose excretion occurred in the acidic range be- tween p H 4.5 and p H 4.8. Quite low rates were observed at p H values >5.4; ^CC?2 fixation, however, extended at reasonable rates up to p H 7.6. A t the higher p H values, maltose was still the dominating compound in the small amount o f excreted material.

Maltose excretion was proportional to illuminance, up to about 3000 lx, whereas ^ C 0 2 fixation in the cells continued in a linear fashion up to 6000 lx (Fig. 4). The percentage of ^ C excreted as maltose, therefore, was higher at the lower illuminances; at 1 700 lx it amounted to 36% of the total fixed *^C. The temperature optimum for [^CJmaltose excretion in the light and for *^C02 assimilation was broad, extending between 28° C and 36° C (data not shown).

E

Q .

4

20 - E

10

pH

Fig. 3. Dependence on p H o f ^ C 0 2 fixation by cells o f (7?/rvf//^

sp. 3.83 a n d o f [ ^ C ] m a l t o s e excretion. * — * , maltose excretion;

A A , ^ C 0 2 fixation. The f o l l o w i n g buffers were used at a con- centration o f 50 m M t h r o u g h o u t : citric acid-trisodium citrate ( p H 3.9-5.4), 2-(N-morpholino)ethanesulfonic a c i d - N a O H ( p H 5.8-7.0), 4-(2-hydroxyethy!)-l-piperazineethanesulfonic acid- N a O H ( p H 7.0-8.2). T h e rate o f maltose excretion at p H 4.8 was 7.8 u m o l - r r * - ( m l p . c ) " *

E

C L

E

Q .

Mtuminance (ktxl

Fig. 4. F i x a t i o n o f ^ C O , and excretion o f [ ^ C ] m a l t o s e by cells of sp. 3.83 in the light as a function o f illuminance.

A — A , in cells; * * . excreted [**T]maltosc. Different i l l u - minances were achieved by calibrated wire screens mounted in front of the vessels, other conditions as given in A^v/<v/<// /;?<'f/;<;</.\

Tabic ! . The effect o f I X C P and o f D F S on ['^( {maltose excretion by cells o f (7?/^//<v sp. 3.81 in the dark. After 1 h o f ' \ ( ) , assimi- lation (see M<7/<v/;// <7/?</ /7;('//?cr/A) the cells were centrifuged. resus- pended in fresh buffer and poison was added in c l h a n o l i c sotution.

to the control on!y ethanol. After shaking for 1 h in the dark in air [ ^ C ] m a l t o s e in the medium was determined. The rates o f maltose excretion arc based on the specific radioactivity o f the

' " C O ,

Poison

F C C P

D F S

Concentration ( M )

0 10 ' 10 '

0 10 ' 5 10 ' 10 ^

[ ^ C ] m a l t o s e excretion (nmol h ' (ml p.c.) ')

4.42 1.11 0.84 4.63 2.92 0.93 0.74

Eb/'rAw^ /^r ;v(7/t^

Sensitivity of maltose excretion against the uncoupler carbonyl cya- nide /7-trif!uoromethoxy phenyl hydrazone ( F C C P ) and the inhibitor dicthylstilbcstrol ( D E S ) would be expected for an active export step. Diethylstilbcstrol has been re- ported to inhibit oxidative phosphorylation and to act on plant plasmalcmma ATPasc (Baikc and Hodges 1977). After assimilation of ^ C 0 2 in the light, the ex- port of [*^C]maltosc was found to be inhibited by F C C P and by D E S (Table 1). These results arc consistent with a mechanism depending on intact p H gradients and-or the presence of A T P . The data could be explained, how- ever, also if energy-requiring steps were involved in the formation of maltose.

Evidence for active transport was derived from mea-

surements of maltose excretion against a concentration

gradient. For this purpose an algal suspension was made

2 m M with respect to maltose at the beginning of an

TaMe 2. Time course of changes in intra- and extracellular sugar concentrations in a cell suspension of C/?/on?//a sp. 3.83. Cells were suspended at a cell density of 100 ul p.c.-ml"* in 50 ml 50 m M citric acid-trisodium citrate buffer (pH 4.8) containing 2 m M mal- tose, and were shaken in a 500-ml Erlenmeyer flask which was continuously gassed with 2% (v/v) C O 2 in air. Illumination was 15000 lx and the temperature 28° C. After different times, 6.5-ml aliquots of the suspension were withdrawn, vacuum-filtered on 0.45-pm cellulose-nitrate membrane filters (5 cm diameter) and washed three times with ice-cold citrate buffer. Extracellular mal- tose was assayed enzymatically in aliquots from the filtrate and intracellular maltose, sucrose and glucose+ glucose-6-phosphate in algal extracts obtained according to Bieleski (1982; see Afaffna/

# H 6 f ;wf/ic&). Intracellular concentrations are corrected for inter-

cellular water content of packed cells Concentration

(mM)

Time (min)

0 30 60 90 120 150 180 Intracellular sugar

Glucose + glucose-6-phosphate Sucrose

Maltose

Extracellular maltose Maltose concentration ratio outside/inside

0.34 0.34 0.32 0.31 0.29 0.40 - 4.38 3.93 3.98 3.96 3.86 4.79 0.43 0.38 0.35 0.45 0.51 0.59 2.17 2.38 2.74 3.20 3.50 3.94 4.57 5.5 7.2 9.2 7.8 7.7 7.8

3 .

E

C L O

E

21 o

20

^ 15

^ 10 o .0 5 o*

0)

experiment and the maltose concentration in the medium was followed for a period of 3 h ; during that time, sam- ples of cells were collected by filtration, extracted and the cellular maltose concentration was determined. Ta- ble 2 shows that the intracellular concentration increased only from 0.43 m M to 0.59 m M , whereas the extracellu- lar maltose concentration rose from 2.2 m M to 4.57 m M . Thus, net maltose transport occurred against a concentration gradient, indicating an active transport.

The reason for the relatively constant concentration ra- tios outside/inside of 7-8 after different times (Table 2) is the more or less parallel increase of intracellular and extracellular maltose concentration. The data, however, do not indicate an equilibrium between efflux and influx:

note that all through the experiment a net efflux took place at a fairly constant rate. Even when the medium was made 5 m M with respect to maltose, net excretion of this sugar was observed (Fig. 5 b). To be sure that the cells had not lost large amounts of maltose by the filtration and washing procedure leading to values of intracellular maltose concentration that were too low, a control experiment was carried out employing the sili- cone-oil centrifugation technique for cell separation (see Werkheiser and Bartley 1957; Ma;eri'<3/ anaf ^ ^ ^ o ^ ) . W i t h both methods, essentially the same intracellular maltose concentration was obtained, e.g. 0.41 m M (sili- cone-oil centrifugation) and 0.48 m M (filtration).

The intracellular concentration of glucose+ glucose- 6-phosphate was found to be comparable to that of mal- tose, whereas sucrose was present at a 10-times higher concentration (4.2 m M ; Table 2). Since, however, no su- crose was encountered in the medium (Fig. 2 a), the mal- tose-export system seems to be quite specific (see Zie- seniB 1982).

Time Ih!

Fig. 5a, b. Kinetics of [^CJmaltose uptake (a) and of maltose ex- cretion (b) by CA/orf//a sp. 3.83. a For uptake measurements, 4.5 ml of algal suspension (50 ul p.c. - m l " * in 50 m M citric acid-trisodium citrate buffer, p H 4.8) were shaken in the dark in a 25-ml Erlen- meyer flask in the presence of [^Cjmaltose (5 m M ) with a specific radioactivity of 658 Bq-umol"* equivalent to 34640 cpm-umol"*.

At different times, 0.5 ml-samples were subjected to vacuum nitra- tions, washed three times with 2 ml ice-cold suspension buffer and the cells' radioactivity was determined (Fischer etal. 1989). At 15 min and 180 min, an aliquot of unflltered suspension was taken for determination of total radioactivity. For calculation of isotopic equilibration the values of packed cells were corrected for 34%

intercellular water, b Excreted maltose was determined enzymati- cally (see M%?^r;'<2/ <37?<^ / ? ? ^ ? / ? 6 ) 6 ^ ) after the cells had been removed by centrifugation; incubation conditions were as in a except that unlabeled maltose was used

If maltose leaves the cell by catalyzed diffusion, one should expect an uptake of external [^CJmaltose. When

tose, less than 20% of isotopic equilibrium was reached in the cells after 3 h and the rate of uptake was only 0.11 nmol - h " * * (ml p.c.)" * as calculated from the specif- ic radioactivity of the [^Cjmaltose (Fig. 5 a). A t the same time, maltose was excreted at a more than 80-fold rate (9.2 umol - h " * - (ml p.c.) * *, Fig. 5 b). Thus, the alga seems to drive a unidirectional maltose transport and is almost inaccessible to this sugar from the outside.

These flux characteristics are generally associated with active transport (Komor 1982).

Glucose was taken up by C/z/o;r//<3 sp. 3.83 at reason-

able rates ( 7 . 3 u m o l - h " * - ( m l p.c.)"* at p H 4 . 8 ,

2 9 i i m o l - h " * - ( m l p.c.)"* at p H 6 . 5 ; data not shown),

whereas sucrose uptake was almost nil.

Discussion

The symbiont from the protozoan ^ r ^ / A ^ r ; \ s//A is able to excrete substantial amounts of maltose, a property which is also exhibited by Chlorellae endosymbiotic with /Vy&a (Muscatine 1965; Cernichiari 1969), P;/ny/7?ff/;//77 (Muscatine e t a l . 1967; Zieseniss etal. 1982; Mucke 1985) and ^ n ^ r (Reisser 1981). The observed rates of maltose excretion (4.0-9.2 u m o l - h " ' - (ml p.c.)"*) are of the same order as the value given by Cernichiari et al.

( 1 . 8 u m o l - h - * - ( m l p.c.)**; 1969). The p H dependence of maltose release with an optimum in the acidic range (Fig. 3) has also been reported for the symbionts of

ra (Muscatine 1965; Cernichiari et al. 1969) and fa/Y/wf-

c;'M?7i

(Zieseniss 1982; Mucke 1985). Glucose excretion

from C/W6)?r//(3 sp. 211-40c, a freshwater sponge sym- biont, displays a similar p H profile (Fischer et al. 1989), which is also indicated for maltose and trehalose release

from

strains isolated from 57(v?^r (Reisser

1981) and

(Pardy etal. 1989), respectively.

This widely distributed characteristic strongly indicates a common mechanism of sugar export in these symbiotic Chlorellae.

Cytological features of CA/oTr/Ay sp. 3.83 (cell size, number of autospores; shape of chloroplast) are well comparable to those of other maltose-excreting algae (Cernichiari e t a l . 1969; Reisser 1984). Besides all the physiological and cytological similarities there are differ- ences in this group of Chlorellae, e.g. in CA/fw//;/ sp.

3.83 photosynthesis and maltose excretion were different in their light-saturation characteristics (Fig. 4) whereas they agreed in the symbiont from //yJ/Y/ (Cernichiari et al. 1969). A nutritional difference is indicated by the ability o f strain 3.83 to grow without added vitamins, whereas isolates from Paramecium require addition of vitamins B i and B ^ (Reisser 1975; Zieseniss et al. 1981;

Mucke 1985).

The sensitivity of maltose excretion to F C C P and D E S points to an energy-requiring step in the transport and-or in the biosynthesis of maltose. The former possi- bility seems to be realized in a Pam/T^c/M/?? symbiont whose maltose excretion was inhibited by vanadate and by D E S with little effect on cellular A T P content and respiration; therefore, these compounds were assumed to act on an energy-transforming plasmalemma ATPasc involved in maltose export (Zieseniss 1982). Net excre- tion of maltose occurred even when this sugar was scven- to eightfold more concentrated in the medium than with- in the cell (Table 2). This finding indicates an active- transport process (see K o m o r 1982). If, however, the cytoplasm from which maltose has to be excreted, com- prises less than 1/7.5^13% of the cell volume (and if all intracellular maltose is located there), a passive efflux depending on, e.g. catalyzed diffusion would be conceiv- able. The volume of the cytoplasm is not known for C/Wor <?//<3 sp. 3.83; a value of 40% can be derived for

C/?/cr<?//<2 u%<:-M3/;;;%

from a study employing quantitative stereology and three-dimensional reconstruction (Atkin- son et al. 1974).

The practically unidirectional movement of maltose into a medium containing 5 m M maltose (ratio of efflux/

influx >80,. see Fig. 5) would require cytosolic concen- trations of maltose much higher than 5 m M if a passive transport were involved; a value of at least 5-80 = 400 m M has to be postulated for the case of unmediated diffusion. A more favored explanation for the predomi- nant maltose efflux is the action of a vectorial active- transport system (see K o m o r 1982).

For Esr/7<'/'/<7?A/ ro//, active uptake of maltose is known in reasonable detail (Wiesmcycr and Cohn 1960;

Szmelcman and Schwartz 1976; Shuman and Treptow 1985). To obtain a closer insight into the mechanism of maltose transport in the symbiotic Chlorellae further experimentation is needed, eventually employing iso- lated plasmalemma vesicles.

This work was supported by the Deutsche ! orschungsgemcin- schaft. T h a n k s are due to D o r i s M e i n d l for skillful experimental help.

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Received 15 December 1989; accepted 23 February 1990