Planta (1995)195:519-524 P l a n t ~

�9 Springer-Verlag 1995

Low-CO2-inducible protein synthesis in the green alga DunMiella tertiolecta Ziyadin Ramazanov 1, Pedro A. Sosa 2, Margaret C. Henk 3, Miguel Jim6nez del Rio 2, Juan Luis Gbmez-Pinchetti 2, Guillermo Garcia Reina 2

Department of Botany, Louisiana State University, Baton Rouge, LA 70803, USA 2 Instituto de Algologia Aplicada, Universidad de Las Palmas, Box 550, Las Palmas, Gran Canaria, Spain 3 Department of Microbiology, Louisiana State University, Baton Rouge, LA 70803, USA

Received: 9 May 1994 / Accepted: 25 July 1994

Abstract. In the green marine alga Dunaliella tertiolecta, a CO2-concentrating mechanism is induced when the cells are grown under low-CO 2 conditions (0.03% CO2). To identify proteins induced under low-CO 2 conditions the cells were labelled with 35SO42-, and seven polypeptides with molecular weights of 45, 47, 49, 55, 60, 68 and 100 kDa were detected. The induction of these polypep- tides was observed when cells grown in high CO 2 (5% CO2 in air) were switched to low CO2, but only while the cultures were growing in light. Immunoblot analysis of total cell protein against pea chloroplastic carbonic an- hydrase polyclonal antibodies showed immunoreactive 30-kDa bands in both high- and low-CO2-grown cells and an aditional 49-kDa band exclusively in low-CO z- grown cells. The 30-kDa protein was shown to be located in the chloroplast. Western blot analysis of the plasma- membrane fraction against corn plasma-membrane AT- Pase polyclonal antibodies showed 60-kDa bands in both high- and low-CO 2 cell types as well as an immunoreac- tive 100-kDa band occurring only in low-CO2-grown cells. These results suggest that there are two distinct forms of both carbonic anhydrase and plasma-mem- brane ATPase, and that one form of each of them can be regulated by the CO2 concentration.

Key words: ATPase - Carbonic anhydrase - CO2-con- centrating mechanism - Dunaliella - Protein synthesis - Photosynthesis

Introduction

In algae, the assimilation of dissolved inorganic carbon (CO2 + HCO3-) (DIC) from the environment is affected by

Abbreviations: CA=carbonic anhydrase; DIC = dissolved inor- ganic carbon (CO2+ HCO3-); CCM = COz-concentrating mecha- nism; low CO 2 = air containing 0.03% CO2; high CO2 = air supple- mented with 5% CO 2 (v/v) Correspondence to: G. Garcia Reina; FAX: 34(28)682830

the COz-concentrating mechanism (CCM), a transport system that enhances the delivery of CO2 to ribulose-l,5- bisphosphate carboxylase/oxygenase (Rubisco) (Badger et al. 1980; Zenvirth and Kaplan 1981; Raven and Lucas 1985; Aizawa and Miyachi 1986; Badger 1987; Sa- muelsson et al. 1990; Moroney and Mason 1991; Spald- ing et al. 1991). The CCM appears to be present in most algal groups, although it has not yet been biochemically characterized.

A CCM is induced in the halotolerant marine unicel- lular alga Dunaliella tertiolecta when it is grown under low-CO 2 conditions (Aizawa and Miyachi 1984, 1986). This increased affinity for CO: facilitates both the active transport of DIC into the cell and the active assimilation of internal carbon. Dunaliella acclimates to low CO 2 con- ditions with an apparent photosynthetic Km (CO2) of < 1-5 gM (Aizawa and Miyachi 1984; Ramazanov and Cardenas 1992).

The enzyme carbonic anhydrase (CA) has been found to play an important role in the CCM and in CO2 assim- ilation in microalgae (Spalding et al. 1983, 1991 ; Aizawa and Miyachi 1984, 1986; Coleman and Grossman 1984; Moroney and Mason 1991; Sfiltemeyer et al. 1990, 1993; Ramazanov and Cardenas 1992). Clarification of the ac- tual role played by CA in the regulation of carbon assim- ilation and in the CCM is complicated due to the exis- tence of several forms of CA (Husic et al. 1989; Fukuzawa et al. 1990; Rawat and Moroney 1991; Ramazanov and Cardenas 1992). These CA isoforms differ not only in their intracellular location, but also in the nature of the dependence of their activity upon the cultivation condi- tions, CO 2 concentration in particular (Pronina et al. 1981; Husic et al. 1989; Fukuzawa et al. 1990; Rama- zanov and Cardenas 1992; Sfiltemeyer et al. 1993). In D. tertiolecta and D. salina, CA has been reported to be located on the cell surface and to increase the affinity for CO 2 during photosynthesis (Aizawa and Miyachi 1984; Booth and Beardall 1991; Goyal et al. 1992; G6mez- Pinchetti et al. 1992; Ramazanov and Cardenas 1992).

However, CA activity alone cannot fully explain the internal inorganic-carbon accumulation that takes place

520 z. Ramazanov et al.: Protein synthesis in Dunaliella

under condi t ions of low external D I C nor the organiza- t ion of the fully implemented C C M in algae (Gehl et al. 1990; Mar t inez et al. 1992). At the same time that Chlamydomonas reinhardtii cells induce the C C M and in- crease their affinity for external inorganic carbon, at least five polypeptides are induced (Coleman and G r o s s m a n 1984; Manue l and M o r o n e y 1988; Spalding and Jeffrey 1989; Spalding et al. 1991 ). These proteins include a 37- k D a periplasmic CA and four other proteins with molec- ular weights of 21, 36, 42 and 44 k D a (Coleman and G r o s s m a n 1984; Manue l and Moroney 1988; Geragh ty et al. 1990; Spalding and Jeffrey 1989; Spalding et al. 1991; R a m a z a n o v et al. 1993).

We have little in fo rmat ion abou t low-CO2-inducible pro te in synthesis in D. tertiolecta. T h i e l m a n n et al. (1992) reported that in D. tertiolecta the 45- and 47-kDa polypept ides induced by low CO2 are const i tuents of the chloroplas t envelope and that they appear to be required to activate the H C O 3 - t ranspor t ing p u m p in these cells. However, the exact funct ional role of these polypeptides in the C C M remains unclear.

The requi rement of a p l a s m a - m e m b r a n e ATPase for active D I C t ranspor t in algae has been suggested (Raven and Lucas 1985; Badger 1987; Th i e lmann et al. 1990; Rota to re et al. 1992; Kar l sson et al. 1994). Several au- thors propose that the low-COz-induced vanada te sensi- tivity in photosynthes is may be caused by the appearance of a vanadate-sens i t ive ATPase involved in HCO3 trans- port (Raven and Lucas 1985; T h i e l m a n n et al. 1990; Kar lsson et al. 1994). The mechan i sm by which such an ATPase may be involved in D I C t ranspor t needs more invest igat ion. To date, none of the low-COz-inducible prote ins in algae has been identified as a H C O 3 trans- port pro te in or as an ATPase. Moreover, it has been suggested that in Dunaliella CO2 is the general form of inorgan ic ca rbon t ranspor ted across the p lasma mem- b rane and that b i ca rbona te enters the cell main ly by an "indirect" mechan i sm after dehydra t ion to CO2 (Aizawa and Miyachi 1986; Booth and Beardall 1991; Rama- zanov and Cfirdenas 1992).

In this work, exper iments with D. tertiolecta cells were carried out to identify prote ins that are preferentially synthesized under low-CO 2 condi t ions.

Materials and methods

Algal culture conditions. Dunaliella tertiolecta wild type, strain 999, was obtained from the UTEX Culture Collection (Texas, USA). Cells were first grown synchronously by using a 12 h light/12 h dark cycle for 3 d at 26_+ I~ After this period, cultures with a chloro- phyll (Chl) concentration of 3 4 lag-ml ~ were switched to continu- ous illumination with white light (300 lamol quanta.m 2.s 1) sup- plied by fluorescent lamps. Under these conditions, the culture starts non-synchronous growth. Thus, non-synchronous cultures have been used for the experiments. The culture concentration of 3~4 lag Chl.ml ~ was maintained by daily dilution with fresh medi- um. The culture medium contained 0.5 M NaC1, 4 mM KNO3, 2 mM MgSO4, 1.9 mM MgC12, 0.01 mM Ca(NO3)2, 4 mM K2HPO 4 and the micronutrient solution as described by Jim6nez del Rio et al. (1994). This medium was buffered at pH 7.5 with 20 mM Tris- HC1. Algae were grown in 0.5-k glass bottles (5 cm in diameter) sparged with either a CO2:air mixture (5:95, v/v; high-CO2 cells) or

with air (0.03% CO2, low-CO2 cells). The gas flow was kept at a high rate (between 1500-2000 mL.min ~) to favour an equilibrium con- centration of DIC (Karlsson et al. 1994).

Photosynthesis assays. Photosynthesis was measured in 2-ml algal samples with a Clark-type oxygen electrode (Hansatech Instru- ments, Norfolk, UK). Algae were centrifuged at 5000.g for 5 min and the pellet (34 lag Chl.ml 1) resuspended in 2 ml of a 20 mM Hepes-KOH buffer supplemented with 0.5 M NaCI (pH 7.3) and transferred to the electrode chamber. Cells were allowed to con- sume the inorganic carbon of the buffer and the intracellular pool of DIC until no net photosynthesis was observed. Bicarbonate was added when net 02 evolution had leveled off. The irradiance was 500 lamol quanta-m 2-s ~.

Carbonic anhydrase activity. The CA activity was determined in cells washed once with 10 mM Na-phosphate (pH 7.5), supplement- ed with 0.5 M NaCI (as an osmotic component). After centrifuga- tion at 5000.g for 5 min, pellets were resuspended in ice-cold CA buffer [30 mM N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid) (EPPS), 1 mM EDTA, 0.1 mM dithiothreitol (DTT) and 1 mM MgSO4, pH 8.2] to a Chl concentration of 10 lag.ml ~. The CA activ- ity was measured in cells disrupted in CA buffer as described previ- ously (Ramazanov and C&rdenas 1992).

Activity of CA was also measured in fractions eluted from agarose affinity columns. Cell homogenates were centrifuged at 15000.g for 5 rain, and supernatants subjected to 35% and 70% ammonium sulfate fractionation. The 70% ammonium sulfate pre- cipitate was dialyzed against 2 L of 100 mM NaC1, 1 mM EDTA, 20 mM sodium phosphate (pH 6.8). The dialysate was centrifuged at 15000.g for 10 min and the supernatant was loaded on a p- aminomethylbenzene sulfonamide-agarose affinity column (Phar- macia, Uppsala, Sweden). The column was washed with 25 mM Tris, 22 mM NazSO 4 (pH 8.2), followed by 25 mM Tris, 300 mM NaC104 (pH 8.7). Elution of the protein fractions was monitored with a UV-detector. Following elution of nonspecifically associated proteins, the column was washed with 100 mM sodium acetate, 500 mM NaCiO4, pH 5.6 (Rawat and Moroney 1991), and eluted protein fractions that showed CA activity were subjected to West- ern blot protein analysis.

Labelling cells with 3~S042 . Protein labelling by 35SO42 was per- formed according to Spalding and Jeffrey (1989). High-CO2-grown cells were switched to minimal media containing 1/10 MgSO 4 con- centration for 2 d. Cells were harvested by centrifugation at 5000-g for 5 min, the pellet washed twice with growth media lacking sul- fate, then centrifuged again. The pellet was resuspended in growth media without sulfate to a Chl concentration of 5 lag.mI 1 and divid- ed among seven 200-ml glass tubes (40 mm in diameter). Tubes were bubbled with air or with air supplemented with 5% CO2. Then 455 kBq of carrier-free H235S04 (370 TBq.mmol ~) was added to the cultures. After 5 h incubation with 35SO42 , cells were harvested by centrifugation at 5000.g for 5 min and the pellet was washed twice with 30 ml 30 mM Hepes-KOH (pH 7.5) supplemented with 0.5 M NaC1, and centrifuged again. The pellet was resuspended in 30 mM Hepes-KOH (pH 7.5) buffer for SDS-PAGE. To compare different treatments, samples were loaded to equal counts (250000 cpm per lane). The amount of radioactivity incorporated into the algal cells was determined by taking aliquots of cells in buffer and counting the sample using a Beckman LS 1801 liquid scintillation counter.

Plasma-membrane isolation. Plasma membranes were isolated ac- cording to the method of Larsson (1985). Cells were broken as described previously by Ramazanov and Cardenas (1992) and the crude homogenate was centrifuged at 1500.g for 30 min to remove chloroplasts and cell debris. The supernatant was collected and centrifuged for 1 h at 28000-g to pellet microsomal membranes. The pellet was resuspended in 10 ml of Solution 1 (330 mM sucrose, 60 mM NaC1, 10 mM DTT, 0.1 mM EDTA, 0.1 mM phenylmethyl- sulfonylfluoride (PMSF) and 5 mM potassium phosphate, pH 7.8; Larsson 1985). This solution was overlaid with 50 g of Solution 2 to

Z. Ramazanov et al.: Protein synthesis in Dunaliella

give a final concentration of 6.5% (w/w) dextran T-500, 6.5% (w/w) polyethylene glycol (PEG) 3350, 330 mM sucrose, 2-5 mM KC1, 10 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF and 5 mM potassium phosphate (pH 7.8) in order to facilitate biphase partition (Widell 1987). The suspension was inverted several times and centrifuged for 5 min at 1500.g to separate the phases. Membranes were then separated by washing each of the two phases two additional times. The final upper phase (U3) was diluted fivefold with Solution 1 and pelleted by centrifugation at 80000-g for 1 h. The final pellet was resuspended in Solution 2 and used for Western blot protein analy- sis.

Chloroplast isolation. Chloroplast isolation is described in detail by Jim6nez del Rio et al. (1994). Briefly, 1 L of exponentially grown high-CO2- and low-CO2-acclimated cells were harvested by cen- trifugation at 500.g for 10 min at room temperature. Cells were washed with 25 mM Hepes-KOH (pH 7.5) supplemented with 0.5 M NaC1 and the pellet resuspended in 30 ml of ice-cold disrup- tion buffer containing 50 mM Hepes-KOH (pH 7.2), 300 mM sor- bitol, 2 mM EDTA, 1 mM MgC12, and 1% bovine serum albumin (BSA). Cells were disrupted according to the procedure of Rama- zanov and Cardenas (1992).

Analytical measurements. Protein concentration was estimated ac- cording to Bradford (1976). Chlorophyll was extracted with abso- lute ethanol and quantified using the absorption coefficient given by Winterman and De Mots (1965).

Other methods. Sodium dodecylsulfate-polyacrylamide gel elec- trophoresis was performed with 12% (w/v) acrylamide concentra- tion and/or gradient gel from 10 to 20% acrylamide concentration (Laemmli 1970). Autoradiography was performed using Kodak X- OMAT film.

Pea chloroplast CA antibodies were obtained from Prof. John Coleman (University of Toronto, Canada), Chlamydomonas rein- hardtii periplasmic CA antibodies were obtained from Dr. James V. Moroney (Louisiana State University, USA), and corn plasma membrane ATPase antibodies were provided by Prof. Leonard T. Robert (University of California, Riverside, USA). The immunoblot assay was performed according to the protocol from Bio-Rad Lab- oratories (Bio-Rad, Richmond, Cal., USA) except that 5% non-fat dry milk was used to block the nitrocellulose. Goat anti-rabbit IgG(H + L) horseradish peroxidase conjugate and HRP Color de- velopment reagent were purchased from Bio-Rad Laboratories.

521

Table 1. Photosynthesis and CA activity in D. tertiolecta grown under high- and l o w - C O 2 conditions. Data are means_+ SD

Growth conditions Ko s(COz) a C A b (gM) (units .mg Chl ~)

5% C O 2 40_+5 30_+ 10 0.03% CO2 3_+ 1 390_+ 20

a Ko.5(CO2 ) is the half-maximal rate of photosynthesis. The rate of photosynthesis was measured in 20 mM Hepes-KOH supplemented with 0.5 M NaC1 (pH 7.3) and an irradiance 500 gmol quanta.m 2 ,S 1 .

b D. tertiolecta cells grown under high CO2 concentrations were switched to low CO2 for 6 h and the total CA activity in cell ho- mogenates was measured

Results

Photosynthesis o fD. tertiolecta. Table 1 shows differences in K0.5(CO2) values for h igh- and l ow-COz-g rown D. terti- olecta. Cells g rown under l o w - C O 2 cond i t ions showed increased affinity for i no rgan ic c a r b o n and requ i red 10- fold less CO2 to a t t a in ha l f -max imal p h o t o s y n t h e t i c ra tes than cells g rown at high CO2 levels. Al l exper imen t s pre- sented here were m o n i t o r e d by this m e t h o d to verify tha t the C C M had ac tua l ly been induced in hea l thy cells un- de r low-CO2 g rowth condi t ions .

Labelling cells with 35S04:. Iden t i f i ca t ion of p ro te ins tha t were preferent ia l ly synthes ized unde r low-CO2 condi - t ions (Fig. 1) showed tha t a t least seven po lypep t i de s with mo lecu l a r weights of approx . 45, 47, 49, 55, 60, 68 a n d 100 were induced by low CO2. These po lypep t i de s were in- duced under low-CO2 condi t ions , bu t on ly in l ight, no t in darkness . Three po lypep t ide s wi th mo lecu l a r masses of 45, 47 and 68 k D a a p p e a r e d in large a m o u n t s in low- CO2-grown cells (Fig. 1A). On the o the r hand , a 2 0 - k D a

Fig. 1A, B. Autoradiographic analysis of 35S-labeled protein pro- duced by D. tertiolecta cells. The labeled cell extracts were subjected to gradient 10-20% SDS-PAGE analysis. A Lane 1, marker en- zymes; lane 2, high-CO2-grown cells; lane 3, low-CO 2 cells in light; lane 4, cells were switched to low CO2 in darkness. B Lane 1, marker enzymes; lanes 2, 3, high-CO2-grown cells; lanes 4, 5, low-CO2- grown cells; lanes 2, 4, low-speed pellets that were obtained after centrifigation of cell homogenates at 5000.g for 10 min; lanes 3, 5, low-speed supernatants that were obtained after centrifigation of cell homogenates at 5000.g for 10 min. The arrows indicate the position of the low-CO2-inducible proteins

p ro t e in is repressed in l o w - C O 2 - a d a p t i n g cells (Fig. 1A, compe re lane 1 a n d 2). The 55-, 68- and 100-kDa p ro te ins were obse rved in the low-speed pel let o b t a i n e d after cen- t r i fuga t ion of the l ow-COz-g rown cell h o m o g e n a t e at 5000.g for 10 rain, while a s t rong 6 0 - k D a b a n d a p p e a r e d in the low-speed s u p e r n a t a n t (Fig. 1 B).

Carbonic anhydrase activity. Table 1 shows C A ac t iv i ty of cells unde r the two g r o w t h condi t ions . There was a signif-

522 Z. Ramazanov et al.: Protein synthesis in Dunaliella

Fig. 2. Immunoblot protein analysis of whole-cell homogenates probed with antibodies raised against pea chloroplast CA. Lane 1, high-CO2-grown cells; lane 2, low-CO2-grown cells; lane 3, pea ex- tract used as a positive control. Each lane contained 100 gg protein

Fig. 4. Immunoblot protein analysis of isolated plasma membranes and lysed chloroplast proteins probed with pea chloroplast CA antiserum. Lane 1, marker enzymes; lane 2, plasma membranes from high-CO2-grown cells; lane 3, lysed chloroplasts from high- CO2-grown cells; lane 4, plasma membranes from low-CO2-grown cells; lane 5, lysed chloroplasts from low-CO2-grown cells. Each lane contained 100 gg of protein

Fig. 3. Immunoblot protein analysis of lysed-chloroplast proteins and the soluble protein fraction eluted from a sulfonamide-agarose affinity column probed with pea chloroplast CA antiserum. Lane 1, lysed chloroplasts from low-CO2-grown cells; lane 2, the soluble protein fraction eluted from a sulfonamide-agarose affinity column. Each lane contained 100 gg of protein

icant increase in CA activity in low-CO2-grown D. terti- olecta cells.

Western blot protein analysis. Figure 2 shows protein im- munoblo t analysis using antibodies raised against pea chloroplastic CA. A band of 30 kDa appears in cells grown under both conditions, and a 49-kDa band specif- ically appears only in low-CO 2 cells.

Immunob lo t analysis of the soluble protein fraction eluted from a sulfonamide-agarose affinity column and of lysed chloroplasts isolated from low-COz-cells showed that pea CA antibodies immunoreacted with a 49-kDa protein from the former and a 30-kDa protein from the latter (Fig. 3).

For the further localization of the 30-kDa protein, chloroplasts and plasma membrane were isolated from both high- and low-CO2-grown cells and probed with antibodies raised against pea chloroplast CA. Figure 4 shows that the 30-kDa protein is located in the chloro- plasts and not in plasma membranes. Similar results were obtained when spinach chloroplast CA antibodies were used (data not shown). An immunoblo t of the D. terti- olecta proteins probed with antibodies raised purified Chlamydomonas reinhardtii periplasmic CA antibodies did not show cross-reactivity with cell homogenates from either high- or low-COz-grown cells (Fig. 5).

Figure 6 shows an immunoblo t analysis of cell plasma membranes from both types of cell probed with antibod-

Fig. 5. Immunoblot protein analysis of D. tertiolecta cell ho- mogenate probed with antibodies raised against Chlamydomonas reinhardtii periplasmic CA. Lane 1, low-CO2-grown C. reinhardtii cells used as a positive control; lane 2, low-CO2-grown D. tertiolecta cells

Fig. 6. Inmunoblot protein analysis of a plasma-membrane fraction from D. tertiolecta probed with antibodies raised against plasma- membrane ATPase. Lane 1, low-CO2-grown cells; lane 2, high-CO 2- grown cells. Each lane contained 100 gg of protein

ies raised against corn plasma membrane ATPase. The ATPase antibodies reacted with the 60-kDa protein in plasma membranes isolated from both cell types and with the 100-kDa protein that appears only in low-CO z- grown cells.

Discussion

The high affinity for DIC shown by D. tertiolecta cells grown in low CO2 indicates that these cells induce the CCM. Since the appearance of the low-COz-inducible

Z. Ramazanov et al.: Protein synthesis in Dunaliella 523

proteins correlates with induction of the CCM (Coleman and Grossman 1984; Manuel and Moroney 1988; Spald- ing and Jeffrey 1989; Spalding et al. 1991), these polypep- tides have been suggested as candidates for involvement in the CCM. Our results show that at least seven polypeptides with molecular weights of 45, 47, 49, 55, 60, 68 and 100 kDa are induced by low CO 2 (Fig. 1). Of these, three polypeptides with molecular masses of 45, 47 and 68 kDa appear in large amounts in l o w - C O 2 cells. The 45-kDa and the 47-kDa polypeptides have been specifi- cally localized to the chloroplast envelope of D. tertiolec- ta cells grown under l o w - C O 2 conditions by Thielmann et al. (1992).

In many microalgae the CCM induced by low CO2 involves both an extracellular and an intracellular CA (Spalding et al. 1983, 1991; Aizawa and Miyachi 1984, 1986; Badger 1987; Siiltemeyer et al. 1990, 1993; Mo- roney and Mason 1991; Ramazanov and Cardenas 1992). Recently, the presence of an extracellular and an intracel- lular CA were also demonstrated in D. tertiolecta cells (Dionisio-Sese and Miyachi 1992; Goyal et al. 1992). In addition, Aizawa and Miyachi (1984, 1986) demonstrated that low-CO2-grown D. tertiolecta have higher CA activ- ity on their surfaces than within.

Our results (Table 1) also show an increase in CA ac- tivity in low-COz-grown cells, coincident with the ap- pearence of a 49-kDa band which reacts with pea chloro- plast CA antibody (Fig. 2). A 49-kDa protein in low-CO 2 D. tertiolecta is also shown to be a soluble protein which binds to the sulfonamide/agarose affinity column, and which is the sole immunoreactant among the soluble proteins eluted from the column (Fig. 3). This 49-kDa soluble protein possibly accounts for the rise in surface (or extracellular) CA activity even though the cross-reac- tivity is against a chloroplast membrane (or intracellular) CA. Clarification of this point is of special interest in order to understand (i) the roles of the different isoforms of CA in the biochemical mechanisms underlying adap- tation of the photosynthesizing cells from high to low CO2 concentrations, and (ii) the functional role of the cell organelles in this process.

The anti-CA reactive 30-kDa band appears to be a chloroplast protein and is present in both high- and low CO2-grown cells (Fig. 3, 4). This protein does not appear in the soluble protein fraction which binds to the sulfon- amide/agarose affinity column, but presumably passes freely through it, being bound to other membrane com- ponents. Siiltemeyer et al. (1993) reported a similar char- acteristic for the chloroplastic CA of Chlamydomonas reinhardtii. When blotted against antibodies to the 37- kDa periplasmic (extracellular) CA of C. reinhardtii (Rawat and Moroney 1991) however, the chloroplast (in- tracellular) 30-kDa protein of D. tertiolecta did not cross- react (Fig. 5). This finding contradicts the results of Goy- al et al. (1992) who reported that antiserum prepared against the 37-kDa polypeptide of periplasmic CA from C. reinhardtii did immunoreact with the 30-kDa polypep- tide of D. tertiolecta, (without reference to the cellular localization of their 30-kDa protein). A possible explana- tion is that antibodies raised in different hosts were used in different experiments. Nevertheless, the immunoreac-

tivity of the pea chloroplast CA antibodies with D. terti- olecta proteins provides evidence worthy of further ex- ploration.

It is generally accepted that green algae are capable of inducing an ATPase-dependent HCO3 transport mecha- nism (Raven and Lucas 1985; Badger 1987; Thielmann et al. 1990; Karlsson et al. 1994). This mechanism is induced by limited CO2 concentrations (Thielmann et al. 1990) and seems to be required for the transport of HCO 3 across plasma membranes and the inner chloroplast membrane (Beardall 1981; Beardall and Raven 1981; Raven and Lucas 1985; Goyal and Tolbert 1989; Thiel- mann et a1.1992). The requirement of a plasma-mem- brane ATPase for DIC transport in algae has been demonstrated in cells growing under low CO 2 concentra- tions (Thielmann et al. 1990; Karlsson et al. 1994), al- though the mechanism by which this ATPase is involved in DIC transport is still unknown. A specific protein for the transport of bicarbonate has not yet been identified, and there is some controversy about the localization of the putative transporter (Marcus et al. 1984; Goyal and Tolbert 1989; Thielmann et al. 1990). Pick (1992) pro- posed that the ATPase in plasma membranes and chloro- plast envelopes is probably not a bicarbonate pump for the following reasons: (i) adaptation to low CO: does not increase the vanadate-sensitive ATPase activity in the plasma membrane, (ii) the ATPase activity in plasma membranes is not affected by bicarbonate, and (iii) ATP- dependent bicarbonate transport in plasma-membrane vesicles could not be demonstrated. In the same study, Pick reported that Dunaliella species contain plasma- membrane ATPases with molecular weights of about 60 and 100 kDa. Our results show that a 100-kDa protein is present among the 35SO42 -labelled low-COe-inducible proteins (Fig. 6), that both a 100-kDa protein and a 60- kDa protein are immunoreact with antibodies raised against purified plasma-membrane ATPase of higher plants, and that both are located in the plasma-mem- brane fraction. This evidence suggests that the constitu- tive 60-kDa and the low-CO2-inducible 100-kDa proteins in D. tertiolecta cells are the same plasma-mem- brane ATPases that have been characterized by Pick (1992). The 60-kDa protein could be the constitutive plasma-membrane ATPase whose activity is changed neither by low-CO 2 adaptation (CCM), nor by varying bicarbonate concentration. The 100-kDa inducible protein, localized in the plasma membrane, also im- munoreacts with corn plasma-membrane ATPase anti- bodies. This protein may be responsible for the vanadate inhibition of photosynthetic HCO 3 transport which is seen only in algae grown under low-CO2 conditions (Thielmann et al. 1990; Karlsson et al. 1994).

In conclusion, our results suggest that both CA and the plasma-membrane ATPase each exist in two forms in D. tertiolecta, that one of each pair is controlled by low CO 2 in the culture medium, and that the 49-kDa and 100-kDa labelled polypeptides from low-CO2-grown cells represent these inducible enzymes.

We thank Prof. John Coleman for providing antibodies raised against pea chloroplast CA, Dr. James V. Moroney for providing

524 Z. Ramazanov et al.: Protein synthesis in Dunaliella

antibodies raised against the 37-kDa periplasmic carbonic anhy- drase of CO2 Chlamydomonas reinhardtii, and Prof. Leonard T. Robert for a gift of corn plasma-membrane 100-kDa ATPase anti- bodies. We thank Dr. Jeanine Olsen (University of Groningen, the Netherlands) for style comments. This work was supported by the Instituto Tecnol6gico de Canarias (Spain).

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