Plant Cell Physiol. 37(4): 431-437 (1996)
JSPP © 1996
Two Polypeptides Inducible by Low Levels of CO2 in Soluble Protein
Fractions from Chlorella regularis Grown at Low or High pH
Akira Satoh ' and Yoshihiro Shiraiwa1'2-3
1
2
Graduate School of Science and Technology, Niigata University, Ikarashi 2-8050, Niigata, 950-21 Japan
Department of Biology, Faculty of Science, Niigata University, Ikarashi 2-8050, Niigata, 950-21 Japan
Previous studies suggested that certain protein(s) other
than carbonic anhydrase might play an important role in
the facilitated transport of dissolved inorganic carbon
(DIC) from the medium to the site of CO2 fixation by
ribulose-l,5-bisphosphate carboxylase/oxygenase in the unicellular green alga Chlorella regularis adapted to lowCO2 (ordinary air) conditions [Shiraiwa et al. (1991) Jpn.
J. Phycol. 39: 355; Satoh and Shiraiwa (1992) Research in
Photosynthesis, Vol. Ill, p. 779]. The proteins that might
be involved in this facilitated transport of DIC were investigated by pulse-labeling of induced proteins with 35S-sulfate
during adaptation of cells grown under high-CO2 conditions to low CO2. Analysis by SDS-PAGE revealed that synthesis of two polypeptides, with molecular masses of 98
and 24 kDa, respectively, was induced under low-CO2 conditions. The 24-kDa polypeptide was induced at pH 5.S but
not at pH 8.0, whereas the 98-kDa polypeptide was induced at both pH 5.5 and pH 8.0. The possible role of
these polypeptides in the facilitated transport of DIC in
Chlorella regularis is discussed.
Key words: Algal photosynthesis — Chlorella regularis —
CO2 pump — Induction of carbonic anhydrase — LowCO2 adaptation — Low CO2-induced polypeptide.
Various unicellular algae and cyanobacteria are able to
induce the facilitated transport of dissolved inorganic
carbon (DIC) from the medium to the CO2-fixation enzyme, Rubisco, when grown under low-CO2 conditions.
This facilitated transport of DIC increases the affinity of the
photosynthetic machinery for DIC, in particular in cells
adapted to the low level of CO2 in ordinary air. The mechAbbreviations: ATPase, adenosine triphosphatase; CA, carbonic anhydrase; CBB, Coomassie brilliant blue; CHI, cycloheximde; DIC, dissolved inorganic carbon; high-CO2 cells, algal cells
grown in air enriched with 1% CO2; K\n (CO2), concentration of
CO2 that yields one-half of the maximum velocity; IOW-CO2 cells,
algal cells grown in ambient air that contained approximately
0.04% CO2; PAGE, polyacrylamide gel electrophoresis; PCV,
packed cell volume; PSL, photo-stimulated luminescence;
Rubisco, ribulose-l,5-bisphosphate carboxylase/oxygenase; Kmax,
maximum velocity.
3
Correspondence should be addressed to Y. Shiraiwa.
anism involves a DIC pump that is located on the plasma
membrane and the chloroplast envelope and/or carbonic
anhydrase (CA) located at the cell surface, in the chloroplasts or in the carboxysomes (for reviews, see Aizawa and
Miyachi 1986, Badger 1987, Badger and Price 1992, Suzuki
et al. 1994).
The physiological functions and molecular properties
of CA have been studied actively by Miyachi's group (for
review, see Suzuki et al. 1994). However, the molecular
properties of the DIC pump remain unclear. In attempts to
identify the polypeptide(s) associated with the CO2-concentrating mechanism, many studies, mainly with Chlamydomonas, have been reported (Coleman et al. 1985, Badour
and Kim 1988, Bailly and Coleman 1988, Manuel and
Moroney 1988, Spalding and Jeffrey 1989, Geraghty et al.
1990, Spalding et al. 1991, Ramazanov et al. 1993), as well
as some studies with Dunaliella tertiolecta (Thielmann et
al. 1992, Ramazanov et al. 1995b).
In Chlorella, little information is available about possible polypeptides that are inducible by low levels of CO2 and
are involved in the CO2-concentrating mechanism. However, the existence of a DIC pump and/or the internal accumulation of DIC has been demonstrated in various algae
(Beardall and Raven 1981, Beardall 1981, Shelp and Canvin 1985, Tsuzuki et al. 1985, Rotatore and Colman 1991,
Satoh and Shiraiwa 1992, Matsuda and Colman 1995).
Ramazanov et al. (1995a) found four low CO2-inducible
polypeptides in Scenedesmus obliquus and six such polypeptides in Chlorella vulgaris (Kosikov). They included
polypeptides that cross-reacted immunologically with antibodies raised against the periplasmic CA of Chlamydomonas reinhardtii. However, it is unclear as yet whether all
of these low CO2-inducible polypeptides are involved in the
CO2-concentrating mechanism.
We have studied the facilitated transport of DIC in
Chlorella regularis and have made the following observation. Chlorella regularis cells absorb free CO2 but not
HCOf ions as the substrate for photosynthesis, irrespective of the concentration of CO2 during adaptation. LowCO2 cells of this alga exhibit higher CA activity, a higher
rate of CO 2 transport and higher photosynthetic affinity for
CO2 than high-CO2 cells. The affinity for CO2 and the rate
of the active transport of CO2 are unaffected by an inhibitor of CA, acetazolamide, but both are decreased greatly
by Na2S, which was reported by Espie et al. (1989) to be in-
431
432
A. Satoh and Y. Shiraiwa
hibitor of the active transport of CO2. These phenomena
were not observed when cells were incubated under lowCO 2 conditions in the presence of cycloheximide. From
these pieces of evidence it was concluded that the contribution of CA to the increase in the affinity of the photosynthetic machinery for CO2 is very small and that some other
protein(s) should, therefore, be involved in the facilitated
transport of DIC in Chlorella regularis (Satoh and Shiraiwa 1992). We also reported that the induction of CA was
suppressed when high-CO2 cells were transferred to air
under acidic pH conditions (Shiraiwa et al. 1991). Since the
molecular properties of CA from Chlorella regularis remain to be denned, identification of the polypeptide(s) associated with the facilitated transport of CO2 might be expected to be easier at a low pH than at a high pH.
In this study, we tried first to confirm that low-CO2
cells adapted at pH 5.5 induce only the DIC-pumping mechanism without CA. Then we attempted to identify a candidate for the protein involved in the DIC pump among the
polypeptides induced by low-CO2 conditions in Chlorella
regularis.
Materials and Methods
Algal material and preculture—The unicellular green alga
Chlorella regularis IAM C-533 was obtained from the Institute of
Applied Microbiology, University of Tokyo. Cells were grown
autotrophically in MC medium (Watanabe 1960). The algal suspension in a 2-liter, flat, oblong glass vessel was continuously
bubbled with ambient air (0.04% CO2) that had been enriched
with 3% CO 2 under continuous illumination with a 200-W incandescent reflector lamp (Toshiba, Tokyo, Japan), as described
previously (Umino et al. 1991). The temperature and the light
intensity were 30°C and 900/*mol m~2s"~', respectively. In the
preculture, the pH of the medium increased from about 6 to about
7. The cells (high-CO2 cells) were harvested by centrifugation
(2,200 xg for lOmin) and used for the following experiments.
Adaptation of cells to various concentrations of CO} and pH
values—High-CO2 cells obtained from the preculture were resuspended in 200 ml of the culture medium that had been buffered
with 50 mM Tris-H2SO4 (pH 8.0) or 50 mM MES-NaOH (pH 5.5)
at a density of 3 ml PCV liter" 1 . The suspensions of cells were incubated in 3% CO 2 in air for 24 h to obtain high-CO2 cells
adapted to pH 8 (H8) or to pH 5.5 (H5.5). The H8-cells and H5.5cells were harvested by centrifugation and each sample was divided in two, and cells were resuspended in fresh culture medium at
pH 8.0 or pH 5.5, as appropriately, at a density of 3 ml PCV
liter"'1. Then bubbling with air was initiated to generate low-CO2
cells. When high-CO2 cells that had been adapted to pH 8 were
transferred to air at pH 5.5, the cells obtained were referred to as
H8/L5.5 cells. In the present study four different samples, namely, H8/L8, H8/L5.5, H5.5/L8 and H5.5/L5.5, were obtained
after 24-h adaptation to low CO2 as shown in Table 1 and Figure
1.
Determination of the rate of the photosynthetic evolution of
O2—The cells were placed in a vessel with a Clark-type O2 electrode (Rank Bros. Co. Ltd, London, England) after resuspension
in a CO2-free buffer, namely, 50 mM HEPES-NaOH (pH 8.0) or
50 mM MES-NaOH (pH 5.5), at a density of 1 ml PCV liter"1.
The rates of the photosynthetic evolution of O2 were determined
at various concentrations of NaHCO 3 to obtain kinetic parameters. The temperature and the light intensity were maintained at
30 c C and 225//molm~ 2 s~ 1 , with illumination from a tungsten
lamp, respectively.
Assay ofCA—Algal cells were disrupted by sonication (three
cycles of sonication for 30 s at a 1-min intervals) by a high-power sonicator (model Bioruptor UCD-200; Cosmobio, Co. Ltd.,
Tokyo, Japan). The resultant cell homogenates were used for assays. The enzymatic activity was determined by measuring the
time required for a change in the pH of 12 mM Veronal buffer
from 8.3 to 7.3 after the addition of a saturating concentration of
CO2 in water by the method of Wilbur and Anderson (1948), as described previously (Umino et al. 1991, Shiraiwa et al. 1991).
Quantitation of protein—Concentrations of protein were determined by the method of Bradford (1976) with a protein assay
kit (Bio-Rad, California, U.S.A.) calibrated with bovine serum
albumin (Sigma, St. Louis, Mo, U.S.A.) as the standard.
Radiolabeling of proteins—Chlorella cells were resuspended
in a sulfate-free medium that contained 50 mM Tricine-NaOH
(pH8.0) or 50 mM MES-NaOH (pH5.5) at a density of 3 ml
PCV liter"1 after cells had been washed three times with the same
medium. The suspension of algal cells (200 ml) was placed in a cylindrical glass vessel at 30°C under illumination by a bank of fluorescent lamps (Aquarium lamps; Hitachi, Tokyo, Japan) at 192
/imolm" 2 s"' from all sides. The suspension was bubbled with
air that contained 3% CO2 for 3 h to obtain cells that were fully
adapted to high-CO2 conditions and to complete the induction of
the system for the active transport of sulfate. To label polypeptides with 35S under high- or low-C0 2 conditions, the bubbling
gas was either unchanged or changed to ordinary air. After a 30min wait to allow the concentration of CO2 to decrease to a steady
level in case of low-CO2 conditions, 100//I of 2 mM [35S]sulfate
(lOMBq ramor') were injected into the medium. The cells were
incubated with [35S]sulfate for 2.5 h and then harvested by centrifugation (2,200 x g for 10 min) after one wash with the same buffer
without [35S]sulfate. The cells were resuspended in 100 mM TrisHC1 (pH 8.3) at a density of 100 ml PCV liter"1 and disrupted by
sonication. Each homogenate was then centrifuged at 195,000 x g
(maximum) for 1 h and the resultant supernatant was subjected to
SDS-PAGE.
SDS-PAGE and autoradiography—SDS-PAGE (10% acrylamide) on a slab gel was performed by the method of Laemmli
(1970) with the Mini-Protean II system (Bio-Rad). Proteins used
as markers were phosphorylase b (94 kDa), bovine serum albumin
(67 kDa), ovalbumin (43 kDa), bovine carbonic anhydrase (30
kDa) and soybean trypsin inhibitor (20.1 kDa). Gels were stained
with Coomassie brilliant blue (CBB) R-250. Then gels were placed
between sheets of cellophane and dried on a gel drier (model 583;
Bio-Rad).
Quantitative analysis of 35S-labeled polypeptides by radioluminography—Radioluminographic analyses were carried out
with a Bio-Imaging Analyzer (model BAS-2000; Fuji Photo Film,
Tokyo, Japan). The dried gel was exposed to the imaging plate for
12 h. The radioactive bands recorded on the imaging plate were
visualized as a radioluminograph on the screen of the image analyzer. Each radioactive band of polypeptide that had been labeled
with 35S in each lane was selected individually, and the intensity of
photo-stimulated luminescence (PSL) was determined in units of
PSL mm" 2 . The lowest value of PSL mm" 2 in a blank area beside
the lanes was defined as the background (BG) and values of
(PSL—BG) mm" 2 were calculated. The radioactivity in each polypeptide band was expressed as the value of (PSL —BG) mm" 2
433
Low CO2-inducible polypeptides in Chlorella
relative to the total value in the respective lane, namely, as the percentage of the total value of (PSL-BG) mm" 2 . The dried gel was
also autoradiographed by exposing it to X-ray film (Medical RX;
Fuji Photo Film) for two weeks at room temperature.
Results
Changes in the kinetic parameters of photosynthesis
and in the activity of CA—High-CO2 cells of Chlorella
regularis had low affinity for CO2. Values of Km (CO2) for
photosynthesis in cells that had been preadapted to pH 8.0
(H8) and to pH 5.5 (H5.5) were 11.9 and 15.7 fiM, respectively, when the values were determined at pH 8.0 (Table
1). Km (COJ of H8-cells and H5.5-cells almost doubled
when it was determined at pH5.5. When high-CO2 cells
were adapted to low (air level; 0.04%) CO2 for 24 h,
K\n (CO2) values decreased markedly under every condition tested. The values of Kxn (COJ determined at pH 5.5
were 2 to 13 times higher than those determined at pH 8.0
(Table 1). Km (COJ was hardly affected by pH during
preadaptation to high-CO2 and during adaptation to lowCO 2 conditions. Vmax of photosynthesis in low-CO2 cells
was about 40% lower than that in high-CO2 cells, irrespective of the pH during the 24-h adaptation (Table 1).
When high-CO2 cells were adapted to low CO2 for 3 h,
the rate of photosynthesis under CO2-limiting conditions increased whereas the rate under CO2-saturating conditions
decreased slightly (Table 2). The ratio of the rate under
CO2-limiting conditions to that under CO2-saturating conditions can be considered to be a parameter of the affinity
of the photosynthetic machinery for CO2. An increase in
this ratio certainly reflects an increase in the affinity of the
photosynthetic machinery for CO 2 . Table 2 shows that the
change in this affinity was suppressed by the addition of
cycloheximide (CHI; lOyUgml"1) during adaptation from
high- to low-CO2 conditions. The inhibition was observed
at both high and low pH. The decrease in the photosynthet-
12
24
36
48
Adaptation period (h)
Fig. 1 Changes in the activities of CA in cell homogenates of
Chlorella regularis during adaptation from high-CO2 (3%) to lowCO2 (ca- 0.04%) conditions at different pH values.
H and L,
periods when cells were kept under high-CO2 and low-CO2 conditions, respectively. O and • , preincubated under 3% CO 2 in air at
pH 8.0 and then transferred to air at pH 8.0 (H8/L8) and pH 5.5
(H8/L5.5), respectively; A and A, preincubated under 3% CO2 in
air at pH 5.5 and then transferred to air at pH 8.0 (H5.5/L8) and
pH 5.5 (H5.5/L5.5), respectively.
ic activity under CO2-saturating conditions during adaptation from high- to low-CO2 conditions required rather
a long time, as reported previously in a cyanobacterium
(Muller et al. 1994). The percent of a decrease in the activ-
Table 1 Effects of the concentration of CO2 and the external pH during 24-h adaptation on the kinetic parameters of
photosynthesis determined at pH 8 and pH 5.5 in C. regularis
Sample
pH during
preadaptation
under 3% CO2
in air
pH during
adaptation
to air
Preculture"
H8
H5.5
8.0
5.5
—
—
—
H8/L8
H8/L5.5
H5.5/L8
H5.5/L5.5
8.0
8.0
5.5
5.5
8.0
5.5
8.0
5.5
—
pH of the medium during preculture under 3% CO2 in air was 7.3.
Kinetic parameters of photosynthesis
K ./2(CO2)
'max
fjumol O21;mlPCV)-'h-']
(MM)
pH8.0
pH8.0
pH5.5
pH5.5
11.9
11.9
15.7
0.42
1.0
0.49
1.9
26.4
35.2
2,400
2,400
2,400
5.3
3.5
5.3
5.3
1,500
1,700
1,800
1,500
2,400
2,500
1,800
1,800
1,800
1,600
A. Satoh and Y. Shiraiwa
434
Table 2 Effects of cycloheximide (CHI; 10^g ml ') on the changes in the affinity of the photosynthetic machinery for
CO2 during adaptation of high-CO2 cells to low-CO2 conditions
1
The rate of photosynthesis' [umol O2 evolved (mlPCVT'rr ]
0.8 mM NaHCO 3
20/iMNaHCO 3
10 mM NaHCO 3
(0.45 [iM CO2)
(18.3 fiMCO2)
(224/iMCO 2 )
(A)
(Q
(B)
Conditions
during adaptation
High CO2 for 24 h
at pH 8.0
at pH 5.5
Low CO2 for 3 h
at pH 8.0
at pH 8.0 with CHI
at pH 5.5
at pH 5.5 with CHI
Ratio
(A)
(A)
129
27
1,334
1,000
2,912
2,312
4.4
1.2
46
43
1,394
95
—
1,040
—
979
2,488
2,770
2,059
2,426
56
3.6
34
2.6
—
38
—
40
706
64
Determined at pH 8.0 and 30°C.
ity was much larger during adaptation for 24 h (Table 1)
than during adaptation for 3 h (Table 2).
The activity of CA in high-CO2 cells was very low, irrespective of pH given during growth (Fig. 1). The activity of
CA increased rapidly when high-CO2 cells were transferred
to low-CO2 conditions at pH 8.0. By contrast, no increase
in the activity of CA was observed when the cells were
adapted to pH 5.5 (Fig. 1).
Low CO2-inducible polypeptides—The soluble polypeptides that were labeled with 35S during adaptation to
high- or low-CO2 conditions at different pH values were analyzed by SDS-PAGE. We analyzed samples that contained
equal amounts of radioactivity (10,000 dpm/lane, Fig. 2A)
or equal amounts (5^/g/lane) of soluble protein (Fig.2B).
B
sup (10,000 dpm / lane)
MW
H8
CBB « S
L8
CBB " S
H5.S
CBB » S
The analysis of the gel in Figure 2A by radioluminography
revealed that polypeptides with molecular masses of 98
and 24 kDa appeared to be strongly labeled with 35S in lowCO2 cells (L8 and L5.5 in Fig. 2A and Table 3), but relatively weakly labeled in high-CO2 cells (H8 and H5.5 in Fig. 2A
and Table 3). The PSL values, corresponding to relative
radioactivities, of the 98-kDa polypeptide in lane L8
(5.5%) and in lane L5.5 (4.6%) were higher than those in
lanes H8 (1.4%) and H5.5 (2.1%), respectively (Table 3).
Thus, this polypeptide appeared to be induced under lowCO2 conditions, irrespective of the pH during adaptation.
By contrast, the PSL value of the 24-kDa polypeptide in
lane L5.5 (22.3%) was higher than those in other lanes
(Table 3). The 24-kDa polypeptide was, therefore, synthe-
sup (5 iig protein/ lane)
L5.5
CBB « S
L8
MW CBB MS
(kDa)
(kDa)
CBB « S
H5.S
CBB
L5.5
CBB " S
(kDa)
Fig. 2 Comparison of the profiles of 35S-labeled soluble proteins from cells of Chlorella regularis that had been adapted to high- and
low-C0 2 conditions at pH 8.0 and pH 5.5.
A: supernatants (sup) with 10,000 dpm of radioactivity were loaded in each lane. B: supernatants containing 5 fig protein were loaded in each lane. H8 and H5.5, cells adapted to high-CO2 conditions at pH 8.0 and pH 5.5, respectively; L8 and L5.5, H8 and H5.5 cells that were subsequently adapted to low-CO2 conditions at pH 8.0 and pH 5.5, respectively.
These cells were the same as the H8/L8 and H5.5/L5.5 cells in Table 1, respectively. CBB and 35S, stained with CBB and the autoradiogram, respectively. MW, molecular mass markers stained with CBB. The molecular masses (kDa) of the marker proteins are indicated
on the left. <, polypeptides that appeared notably at respective lanes. The molecular masses (kDa) of these polypeptides are indicated on
the right.
Low CO2-inducible polypeptides in Chlorella
435
Table 3 Photo-stimulated luminescence (PSL) values, which correspond to radioactivity, for
separated by SDS-PAGE
Sample
(PSL-BG) per mm2 {% of total value)
Polypeptides with molecular masses (kDa) of
58
54
35-37 33 .5-34 .5 32
50
40
38
3S
S-labeled polypeptides
24
Other
regions
3.0
10.2
21.1
100
5.0
4.1
12.1
20.1
100
8.0
11.9
3.4
9.6
15.1
100
6.6
14.6
1.9
22.3
8.5
100
98
69
H8
1.4
3.5
11.9
6.5
8.2
8.3
10.7
8.1
7.1
L8
5.5
1.8
12.0
6.1
9.3
5.6
10.4
8.0
H5.5
2.1
4.4
4.6
5.5
13.9
7.1
14.4
L5.5
4.6
3.9
4.7
5.4
10.3
7.4
9.8
The gel in Figure 2A was analyzed by radioluminography, as described in Materials and Methods. PSL —BG indicates the PSL value of
each band minus that of the background (BG). Values underlined are distinctive under each condition.
sized at an elevated rate under low-C0 2 conditions at pH
5.5.
Other polypeptides—Two other polypeptides, with
molecular masses of 58 and 34 kDa, respectively, were
strongly labeled with 35S at pH 8 (H8 and L8 in Fig. 2 and
Table 3) and at pH 5.5 (H5.5 and L5.5 in Fig. 2 and Table
3).
Discussion
The affinity of the photosynthetic machinery for CO2
increased during the adaptation from high- to low-CO2 conditions in Chlorella regularis, irrespective of the pH of the
medium (Table 1,2). The activity of CA also increased at
pH 8.0 but not at pH 5.5 (Fig. 1). These increases were
blocked by the addition of cycloheximide during adaptation to low CO2 (Table 2). As mentioned in the Introduction, the contribution of CA to the high affinity of the photosynthetic machinery for CO2 in low-CO2 cells seemed to
be very small since the affinity was hardly affected by inhibitors of CA (Satoh and Shiraiwa 1992) or by the suppression
of the induction of CA at acidic pH (Table 1, Fig. 1). From
these pieces of evidence, we assumed that some protein(s)
in addition to CA might be involved in the mechanism of
the active transport of CO2. The function and molecular
properties of CA in this alga are currently being investigated.
Radiolabeling experiments with 35S showed that a 24kDa polypeptide was synthesized at an elevated rate under
low-CO2 conditions at pH 5.5 (Fig. 2, Table 3). The level of
the polypeptide seemed to be very low since it was hardly
detected by staining with CBB even though the radiolabeling was relatively effective (Fig. 2, Table 3). The rapid increase in 35S-labeling of the polypeptide within 3 h (Fig. 2)
parallelled that in the affinity of the photosynthetic machinery for CO2 (Table 2).
Another polypeptide that was induced under low-CO2
conditions had a molecular mass of 98 kDa and was
induced at both pH 5.5 and pH 8.0 (Fig. 2, Table 3). Al-
though the extent of the incorporation of 35S into the 98kDa polypeptide was lower than that into the 24-kDa polypeptide, the 98-kDa polypeptide might have some role in
the facilitated transport of DIC at both pH 5.5 and pH 8.0.
Since the change in the photosynthetic affinity seems to be
related to a change in the active transport of CO2 (see Introduction), it is likely that these two polypeptides are associated with the system for the active transport of CO 2 .
These two polypeptides might be soluble. However, the
possibility that they are extrinsic proteins that are weakly
bound to the membrane cannot be excluded because the
soluble fraction was obtained after strong sonication,
which is neccessary to disrupt the cell walls of Chlorella.
No additional low CO2-inducible polypeptides were found
in membrane fractions of this alga (data not shown). Therefore, it is still unclear whether the polypeptides are the CO2transporter itself, located in the plasma membrane, or
whether they are factors in the soluble fraction that regulate the activity of the CO2-transporter. Further studies are
needed to clarify the function of the two polypeptides in
the facilitated transport of DIC.
In the present study, the CA-protein was not identified
in the analysis by SDS-PAGE of cells adapted to low CO2
at pH 8.0. The CA protein might migrate together with
certain major polypeptide during SDS-PAGE or its level
might be too low for detection on the gel, even though CA
activity was high. In addition, it is still unclear whether the
alga that contains a high COyinducible CA, which was
reported by Tachiki et al. (1992) as the CA2 isozyme
in Chlamydomonas, even though low-level activity of CA
was also detected in high-CO2 cells of Chlorella regularis
(Fig. 1).
In addition to the two low CO2-inducible polypeptides
(98 and 24 kDa), an alkali-specific (58 kDa) and an acidspecific (34 kDa) polypeptide were also found in soluble
fractions of Chlorella regularis (Fig. 2, Table 3). The physiological functions of these polypeptides remains to be determined.
In Chlamydomonas reinhardtii at least five low CO2-in-
A. Satoh and Y. Shiraiwa
436
ducible polypeptides are thought to be involved in the DICconcentrating mechanism. They are a 37-kDa periplasmic
CA (Coleman et al. 1984), two soluble proteins with molecular masses of 46 and 44 kDa (Manuel and Moroney 1988,
Spalding and Jeffrey 1989, Spalding et al. 1991) and two
membrane-associated proteins with molecular masses of
36 and 21 kDa (Spalding and Jeffrey 1989, Geraghty et al.
1990). The 36-kDa polypeptide has been well characterized
and was shown recently to be localized on the chloroplast
envelope (Ramazanov et al. 1993). In Chlorella regularis,
no low CO2-induced, 35S-labeled polypeptides with these
molecular masses were found. This result suggests that the
proteins that are associated with the facilitated transport of
DIC might differ among algal species.
In Scenedesmus obliquus, Thielmann et al. (1990) reported that the ATPase-linked transport of HCO3~, as
well as the transport of CO2, was induced in cells adapted
to air in medium above pH 9. A low CO2-inducible protein
with a molecular mass of 100 kDa, which cross-reacted
with a polyclonal antibody raised against the plasma membrane ATPase of Zea mays, was found in Dunaliella tertiolecta (Ramazanov et al. 1995b). This protein was assumed to be involved in the control of the ATPase-dependent
transport of DIC (Goyal and Tolbert 1989). Although our
98-kDa polypeptide is very similar to the 100-kDa polypeptide in terms of molecular mass, its function might
differ from that of the ATPase since two inhibitors of ATPase, namely, vanadate and diethylstilbestrol, did not. affect
the Kxn (CO2) of photosynthesis measured at pH 8.5 in
IOW-CO2 cells of Chlorella regularis (Satoh and Shiraiwa
1992).
In conclusion, polypeptides of 98 kDa and 24 kDa
were identified as polypeptides that are induced under lowCO2 conditions, when the active transport of CO2 was induced in Chlorella regularis. Further characterization of
these polypeptides is required to clarify their roles in the uptake of DIC under CO2-limiting conditions.
Analysis with the Bio-Imaging Analyzer (model BAS-2000)
was performed with the gracious help of Dr. R. Kuwano of the
Research Laboratory for Molecular Genetics, Niigata University.
The authors are grateful to Prof. H. Takeda for his encouragement and his comments on the original manuscript, as well as to
anonymous reviewers for their stimulating and critical comments.
This work was supported in part by Grants-in-Aid for Scientific
Research on Priority Areas (no. 04273102) and on General
Research Areas (no. 04640622) from the Ministry of Education,
Science and Culture, Japan.
References
Aizawa, K. and Miyachi, S. (1986) Carbonic anhydrase and CO2 concentrating mechanisms in microalgae and cyanobacteria. FEMS Microbiol.
Rev. 39: 215-233.
Badger, M.R. (1987) The CO2-concentrating mechanism in aquatic phototrophs. In The Biochemistry of Plants. A Comprehensive Treatise. Vol.
10, Photosynthesis. Edited by Hatch, M.D. and Boardman, N.K. pp.
219-274. Academic Press, New York.
Badger, M.R. and Price, G.D. (1992) The CO2 concentrating mechanism in
cyanobacteria and microalgae. Physiol. Plant. 84: 606-615.
Badour, S.S. and Kim, W.K. (1988) Detection of specific polypeptides in
Chlamydomonas segnis adapted to atmospheric concentrations of CO2,
using a zwitterionic detergent. Can. J. Bot. 66: 1750-1754.
Bailly, J. and Coleman, J.R. (1988) Effect of CO2 concentration on protein
biosynthesis and carbonic anhydrase expression in Chlamydomonas reinhardtii. Plant Physiol. 87: 833-840.
Beardall, J. (1981) CO2 accumulation by Chlorella saccharophila (Chlorophyceae) at low external pH: evidence for active transport of inorganic
carbon at the chloroplast envelope. J. Phycol. 17: 371-373.
Beardall, J. and Raven, J.A. (1981) Transport of inorganic carbon and the
'CO 2 concentrating mechanism' in Chlorella emersonii (Chlorophyceae).
J. Phycol. 17: 134-141.
Bradford, M. (1976) A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal. Biochem. 72: 248-253.
Coleman, J.R., Berry, J.A., Togasaki, R.K. and Grossman, A.R. (1984)
Identification of extracellular carbonic anhydrase of Chlamydomonas
reinhardtii. Plant Physiol. 76: 472-477.
Coleman, J.R., Green, L.S., Berry, J.A., Togasaki, R.K. and Grossman,
A.R. (1985) Adaptation of Chlamydomonas reinhardtii to air levels of
CO2 and the induction of carbonic anhydrase activity. In Inorganic Carbon Uptake by Aquatic Photosynthetic Organisms. Edited by Lucas,
W. J. and Berry, J.A. pp. 339-359. The American Society of Plant Physiologists, Rockville, MD., U.S.A.
Espie, G.S., Miller, A.G. and Canvin, D.T. (1989) Selective and reversible
inhibition of active CO2 transport by hydrogen sulfide in a cyanobacterium. Plant Physiol. 91: 387-394.
Geraghty, A.M., Anderson, J.C. and Spalding, M.H. (1990) A 36 kilodalton limiting-CO2 induced polypeptide of Chlamydomonas is distinct
from the 37 kilodalton periplasmic carbonic anhydrase. Plant Physiol.
93: 116-121.
Goyal, A. and Tolbert, N.E. (1989) Uptake of inorganic carbon by isolated
chloroplasts from air-adapted Dunaliella. Plant Physiol. 89: 1264-1269.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 227: 680-685.
Manuel, L.J. and Moroney, J.V. (1988) Inorganic carbon accumulation by
Chlamydomonas reinhardtii. New proteins are made during adaptation
to low CO2. Plant Physiol. 88: 491-496.
Matsuda, Y. and Colman, B. (1995) Induction of CO2 and bicarbonate
transport in the green alga Chlorella ellipsoidea. II. Evidence for induction in response to external CO2 concentration. Plant Physiol. 108: 253260.
Muller, C , Tsuzuki, M., Shiraiwa, Y. and Senger, H. (1994) Carbon affinity adaptation of Synechococcus and its phycocyanin mutant to various
CO2 concentrations and light intensities. / . Photochem. Photobiol. 26:
97-101.
Ramazanov, Z., Mason, C.B., Geraghty, A.M., Spalding, M.H. and
Moroney, J.V. (1993) The low CO2-inducible 36-kilodalton protein is localized to the chloroplast envelope of Chlamydomonas reinhardtii.
Plant Physiol. 101: 1195-1199.
Ramazanov, Z., Shiraiwa, Y., Jimenez del Rio, M. and Rubio, J. (1995a)
Effect of external CO2 concentrations on protein synthesis in the green
algae Scenedesmus obliquus (Turp.) Kiitz and Chlorella vulgaris
(Kosikov). Planta 197: 272-277.
Ramazanov, Z., Sosa, P.A., Henk, M.C., Jimenez del Rio, M., G6mezPinchetti, J.L. and Garcia Reina, G. (1995b) Low-CO2-inducible protein
synthesis in the green alga Dunaliella tertiolecta. Planta 195: 519-524.
Rotatore, C. and Colman, B. (1991) The acquisition and accumulation of
inorganic carbon by the unicellular green alga Chlorella ellipsoidea.
Plant Cell Environ. 14: 377-382.
Satoh, A. and Shiraiwa, Y. (1992) Characterization of active transport of
CO2 during photosynthesis in Chlorella regularis. In Research in Photosynthesis, Vol. III. Edited by Murata, N. pp. 779-782. Kluwer Academic
Publishers, Dordrecht, The Netherlands.
Shelp, B.J. and Canvin, D.T. (1985) Inorganic carbon accumulation and
photosynthesis by Chlorella pyrenoidosa. Can. J. Bot. 63: 1249-1254.
Low CC>2-inducible polypeptides in Chlorella
Shiraiwa, Y.t Yokoyama, S. and Satoh, A. (1991) pH-dependent regulation of carbonic anhydrase induction and change in photosynthesis during adaptation of Chlorella cells to low CO2. Jpn. J. Phycol. (S6rui) 39:
355-362.
Spalding, M.H. and Jeffrey, M. (1989) Membrane-associated polypeptides
induced in Chlamydomonas by limiting CO2 concentrations. Plant
Physiol. 89: 133-137.
Spalding, M.H., Winder, T.L., Anderson, J.C., Geraghty, A.M. and
Marek, L.F. (1991) Changes in protein and gene expression during induction of the CCh-concentrating mechanism in wild-type and mutant Chlamydomonas. Can. J. Bot. 69: 1008-1016.
Suzuki, E., Shiraiwa, Y. and Miyachi, S. (1994) The cellular and molecular
aspects of carbonic anhydrase in photosynthetic microorganisms. In Progress in Phycological Research, Vol. 10. Edited by Round, F.E. and
Chapman, D.J. pp. 1-54. Biopress Ltd., Bristol, England.
Tachiki, A., Fukuzawa, H. and Miyachi, S. (1992) Characterization of carbonic anhydrase isozyme CA2, which is the CAH2 gene product, in Chlamydomonas reinhardtii. Biosci. Biotech. Biochem. 56: 794-798.
Thielmann, J., Goyal, A. and Tolbert, N.E. (1992) Two polypeptides in
437
the inner chloroplast envelope of Dunaliella tertiolecta induced by low
CO2. Plant Physiol. 100: 2113-2115.
Thielmann, J., Tolbert, N.E., Goyal, A. and Senger, H. (1990) Two systems for concentrating CO2 and bicarbonate during photosynthesis by
Scenedesmus. Plant Physiol. 92: 622-629.
Tsuzuki, M., Miyachi, S. and Berry, J.A. (1985) Intracellular accumulation of inorganic carbon and its active species taken up by Chlorella
vulgaris 1 lh. In Inorganic Carbon Uptake by Aquatic Photosynthetic Organisms. Edited by Lucas, W.J. and Berry, J.A. pp. 53-66. The
American Society of Plant Physiologists, Rockville, MD., U.S.A.
Umino, Y., Satoh, A. and Shiraiwa, Y. (1991) Factors controlling induction of external carbonic anhydrase and change in K\n (CO2) of photosynthesis in Chlorella regularis. Plant Cell Physiol. 32: 379-384.
Watanabe, A. (1960) List of algal strains in collection at the Institute of Applied Microbiology, University of Tokyo. J. Gen. Appl. Microbiol. 6:
283-292.
Wilbur, K.M. and Anderson, N.G. (1948) Electrometric and colorimetric
determination of carbonic anhydrase. J. Biol. Chem. 176: 147-154.
(Received October 30, 1995; Accepted February 26, 1996)