gation (2000 rpm, Sorvall GSA or GS3 rotor, 10 min), resus- pended in buffer A (0.3 M sucrose, 10 mm EDTA, 10 mM. ACA, 2 mM BAM, 10 mM Tris-HCl, pH 7.5), ...
Plant Physiol. (1989) 89, 133-137 0032-0889/89/89/01 33/05/$01 .00/00
Received for publication May 9, 1988 and in revised form August 20, 1988
Membrane-Associated Polypeptides Induced in Chiamydomonas by Limiting CO2 Concentrations1 Martin H. Spalding* and Martha Jeffrey Department of Botany, Iowa State University, Ames, Iowa 50011 ABSTRACT
acterized which is apparently unable to accumulate inorganic carbon to any substantial extent (18). Although this mutant can be considered to be a putative transport mutant, so supports the inference that inorganic carbon transport is involved, the evidence is not conclusive. Direct physical evidence for a protein or proteins involved in transport of inorganic carbon is lacking, Activity of the CO2-concentrating system is under environmental control. Cells grown at limiting CO2 concentrations (air-adapted) show inorganic carbon transport activity but cells grown at elevated CO2 concentrations (CO2-enriched) do not. We have identified four membrane-associated polypeptides which appear or increase in abundance during adaptation to limiting CO2 concentrations. The appearance of two of the polypeptides coincides with induction of the CO2concentrating system following exposure of the cells to limiting CO2 concentrations. One or more of these polypeptides may be candidates for direct involvement in inorganic carbon
Chiamydomonas reinhardtii and other unicellular green algae have a high apparent affinity for C02, little O2 inhibition of photosynthesis, and reduced photorespiration. These characteristics result from operation of a C02-concentrating system. The C02concentrating system involves active inorganic carbon transport and is under environmental control. Cells grown at limiting CO2 concentrations have inorganic carbon transport activity, but cells grown at 5% CO2 do not. Four membrane-associated polypeptides (Mr 19, 21, 35, and 36 kilodaltons) have been identified which either appear or increase in abundance during adaptation to limiting CO2 concentrations. The appearance of two of the polypeptides occurs over roughly the same time course as the appearance of the C02-concentrating system activity in response to CO2 limitation.
transport.
By comparison with higher plants which use the C3 photosynthetic pathway, Chlamydomonas reinhardtii and other unicellular green algae exhibit unusual photosynthetic characteristics. These characteristics include a very high apparent affinity for C02, little inhibition of photosynthesis by 21%
MATERIALS AND METHODS Algal Strains and Culture Conditions
02, and much reduced photorespiration (5). These character-
istics apparently result from the operation of a C02-concentrating system that increases the intracellular CO2 concentration to a level which suppresses photorespiration by competitive inhibition of RuBP2 oxygenase (1). The mechanism responsible for this effect requires energy, accumulates intracellular inorganic carbon against a very large concentration gradient, and includes carbonic anhydrase (EC 4.2.1.1) as an essential component of the system (1, 4, 17, 20, 22). A saturable component in the kinetics of inorganic carbon uptake has been demonstrated for Chlamydomonas (19), and an electrogenic process has been implicated in the inorganic carbon uptake of Chlorella (4). These points strongly suggest that the C02-concentrating system of unicellular algae involves active transport of inorganic carbon, probably across either the plasma membrane or the chloroplast envelope. Evidence has been presented to support either location of the transporter (3, 11-13, 16), so the actual location is still controversial. A mutant of Chiamydomonas has been isolated and char-
Chlamydomonas reinhardtii cell wall-less strain CW- 15 mt+ (obtained from Dr. R. Togasaki, Indiana University) was cultured axenically in the minimal salts medium described by Winder and Spalding (21) with the exception that the pH of the MOPS-Tris buffer was 7.6 for C02-enriched cells and 7.1 for air-adapted cells. Both cell types were grown in liquid medium on a gyratory shaker (175 rpm). In addition, the C02-enriched cells were constantly aerated with 5% CO2 in air. The pH of both algal cultures during growth was 7.0 to 7.1. All experiments were performed with cells in midphase exponential growth. Induction Experiments
For induction time course experiments all cells, except airadapted controls, were initially grown with CO2 enrichment. The cells were collected by centrifugation, resuspended in sulfate-free minimal salts medium with the appropriate pH buffer, and grown either with 5% CO2 aeration (CO2-enriched controls) or without aeration (air induction and air-adapted controls). To avoid any exposure to limiting C02, the medium for resuspension of the C02-enriched controls was preaerated with 5% CO2. Carrier-free [35S]sulfate (1 mCi/L) was added to the cultures at time intervals and cells harvested 30 min after introduction of the label.
This research was supported by National Science Foundation grant DMB-8500835.
2Abbreviations: RuBP, ribulose-1,5-bisphosphate; ACA, e-aminon-caproic acid; BAM, benzamidine; IEF, isoelectric focusing; MOPS, 3-(N-morpholino)propanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; Rubisco, ribulose-1,5-bisphosphate carboxylase/ oxygenase.
133
134
SPALDING AND JEFFREY
Cell Fractionations and Electrophoresis Cells from 800 mL of culture were harvested by centrifugation (2000 rpm, Sorvall GSA or GS3 rotor, 10 min), resuspended in buffer A (0.3 M sucrose, 10 mm EDTA, 10 mM ACA, 2 mM BAM, 10 mM Tris-HCl, pH 7.5), collected again by centrifugation (5000 rpm, Sorvall SS-34 rotor, 5 min), and resuspended in 5 mL of buffer A plus 0.125 mL PMSF (40 mM in isopropanol). The cells were homogenized thoroughly (750 rpm, 50-100 passes) in a Potter-Elvehjem tissue homogenizer with a motor-driven Teflon pestle. The cell homogenates were centrifuged (2000 rpm, SS-34 rotor, 10 min) to remove unbroken cells and starch grains. The supernatant was recentrifuged (5000 rpm, SS-34 rotor, 30 min) to collect a low-speed membrane pellet. The resulting supernatant was again centrifuged (19,000 rpm, SS-34 rotor, 30 min) to collect a high-speed membrane pellet and a soluble protein fraction. The above procedures were all performed at 4°C. SDS polyacrylamide gel electrophoresis was performed using the method of Laemmli (10, 14) on 10 to 18% acrylamide gradient gels at room temperature. The gels were either silverstained (9) for protein or impregnated with PPO (15), dried and used for fluorographic detection of labeled polypeptides. Molecular size markers used for silver-stained gels were ,3galactosidase (116 kD), phosphorylase b (97 kD), BSA (68 kD), glutamate dehydrogenase (53 kD), ovalbumin (43 kD), glyceraldehyde-3-phosphate dehydrogenase (36 kD), carbonic anhydrase (29 kD), soybean trypsin inhibitor (20 kD), and Cyt c (12 kD). The '4C-labeled molecular size markers (Bethesda Research Labs) for fluorography of 35S-labeled gels were myosin (200 kD), phosphorylase b (97 kD), BSA (68 kD), ovalbumin (43 kD), a-chymotrypsinogen (26 kD), ,Blactoglobulin (18 kD), and lysozyme (14 kD).
Assays of Photosynthesis and Intemal Inorganic Carbon Accumulation
Inorganic carbon accumulation was determined as previously described (1, 19). Cells were exposed to NaH'4CO2 (60 AM, pH 7.0) for 30 s prior to centrifugation through silicone oil. Photosynthetic rate was estimated simultaneously from the acid-stable 14C in the pellet following centrifugation of the cells through silicone oil. RESULTS Observation of the polypeptide profiles of total cell extracts from C02-enriched and air-adapted Chlamydomonas cells revealed no obvious differences between the two when compared by SDS-PAGE (Fig. 1). There also were no obvious differences in the polypeptide profiles of the soluble protein fraction from the two cell types (Fig. 1 ). However, when lowspeed and high-speed membrane fractions from the two cell types were compared, four membrane-associated polypeptides were apparent in the membrane fractions of the air-adapted cells which were either absent or present in very reduced amounts in the same fractions from C02-enriched cells (Fig. 1). These included two polypeptides of approximately 35 and 36 kD, one polypeptide of approximately 21 kD, and one polypeptide of approximately 19 kD Mr. Appearance of the 19 and 35 kD polypeptides was somewhat variable, although it is possible they were not always completely resolved from
Plant Physiol. Vol. 89,1989
other polypeptides of similar size. The two polypeptides at 35 and 36 kD and the 19 kD polypeptide appeared to be more abundant in the low-speed membrane fraction, while the 21 kD polypeptide appeared more abundant in the high-speed membrane fraction. Because of a substantial presence of thylakoid membranes in both membrane fractions, purified thylakoid membranes were prepared as described by Greer et al. (8) from C02enriched and air-adapted cells and their polypeptide profiles compared, by SDS-PAGE, with each other and with the crude, low-speed membrane fraction shown in Figure 1. The 19 and 21 kD polypeptides were absent from the purified thylakoids. There appeared to be some slight retention of the 35 and 36 kD polypeptides in the purified thylakoids from the airadapted cells, but the relative abundance was much decreased compared with the crude membrane fraction (not shown). This indicates that it is unlikely any of the four air-specific polypeptides are resident in the thylakoid membranes. Appearance of newly synthesized polypeptides following exposure of C02-enriched cells to limiting CO2 was investigated using 35S-labeling (Fig. 2). Using this method, a 21 kD polypeptide was apparent in the whole-cell profile 2 h following transfer to limiting CO2. In addition, two new polypeptides at about 45 to 50 kD appeared to be heavily labeled in both the whole-cell and soluble-protein profiles following exposure to limiting CO2, and the large and small subunits of Rubisco exhibited reduced relative labeling in both profiles. In the low-speed membrane fraction, labeling of the 21 and the 36 kD polypeptides were apparent 2 h following transfer to limiting CO2. The other two air-specific polypeptides were not readily observed at 2 h following transfer but, because of the lower resolution afforded by fluorography, the 35 kD polypeptide might have been obscured by the larger one. A similar pattern was observed in the high-speed membrane fraction. Since all four polypeptides were present in the low-speed membrane fraction, a more detailed 35S-labeling time course was performed to further investigate the appearance of newly synthesized polypeptides in this fraction following transfer of C02-enriched cells to limiting CO2 (Fig. 3). The labeled polypeptides at 21 and 36 kD were apparent on the fluorograph by only 1 h following transfer and were prominent thereafter. When the low-speed membrane fractions from the same time course were separated by SDS-PAGE and silver-stained, all four of the air-specific polypeptides appeared over the 8-h time course (Fig. 4). In agreement with the fluorograph in Figure 4, traces ofthe 21 and the 36 kD polypeptides appeared after only 2 h of limiting C02, and the two polypeptides were quite apparent after 4 h and remained prominent thereafter. The 19 and the 35 kD polypeptides were observed after 4 h in limiting CO2 but were not present in substantial amounts even after 8 h. For purposes of comparison with the appearance of airspecific polypeptides, a time course was performed for increase in photosynthetic rate at limiting CO2 and capacity for inorganic carbon accumulation in C02-enriched Chlamydomonas cells exposed to limiting CO2 (Fig. 5). Both were found to increase slightly during the first hour, to increase substantially between 1 and 8 h and to reach by 8 h nearly the level observed at 20 h following transfer to limiting CO2. These
*sVr..oe_Wit1aX.,:,;i4-|_Fil>.F]@.s*,*|,r.l:eU'ht_,'.jS^:-ki;ES[.wE
LIMITING-CO2 INDUCED POLYPEPTIDES IN CHLAMYDOMONAS
1
3 4
2
! 1M
F:5
t.xs .:
11 6 97
_k
68
8 9
6 7
5
.,
5, § i
':
ie.
>4 ^
zs
iI
.. .. X .: :'sii i
" .... .!Qv
. A. rov_ ;.;> |
.
4P
j
4r.. -MV
i
.K
.. .;z
M."Zia.
53 4-. I XI 43 _
'pP
-:,--
imm .f..
*--'-
AP
36
.- 14-:1
z.f
-b. 'MM 4.4
29
am _
135
I
i
Figure 1. Silver-stained SDS-PAGE gel of fractions from C02-enriched (lanes 2, 4, 6, and 8) and air adapted (lanes 3, 5, 7, and 9) Ch. reinhardtii cells. The fractions are: total protein (lanes 2 and 3), lowspeed membrane fraction (lanes 4 and 5), high-speed membrane fraction (lanes 6 and 7), and soluble proteins (lanes 8 and 9). Lane one contains molecular size markers. Arrowheads indicate polypeptides apparently specific for air-adapted cell fractions.
..WI-.
4w
20 12
1
3 4 5 6 7 8 9 10 11 12 13 1415
2
I
p
%I
#r
lb
top
200
t#
97_ 68
4
26
_4*Ls g +
*l 3 *
!w"w-w
w-
*0
Figure 2. Fluorograph of SDS-PAGE gel of 35Slabeled fractions from C02-enriched (lanes 3, 6, 9, and 12), air-adapted (lanes 5, 8, 11, and 14), and C02-enriched Ch. reinhardtii cells exposed to limiting CO2 for 2 h (lanes 4, 7, 10, and 13). Labeled polypeptide pattems are shown for total cell protein (lanes 3, 4, and 5), low-speed membrane fractions (lanes 6, 7, and 8), high-speed membrane fractions (lanes 9, 10, and 11) and soluble proteins (lanes 12, 13, and 14). Lanes 1, 2, and 15 contain 14C-labeled molecular size markers. Arrowheads in lanes 3 and 14 indicate the large and small subunits of Rubisco. Arrowheads in other lanes indicate polypeptides apparently induced by limiting CO2.
18 X ^ 14 increases correlated well with the appearance of the 21 and 36 kD polypeptides. DISCUSSION The work described in this paper has demonstrated the presence of four membrane-associated polypeptides of 19, 21,
35, and 36 kD apparent Mr specific to air-adapted Chlamydomonas reinhardtii cells when compared with C02-enriched cells. Although not demonstrating involvement in the microalgal C02-concentrating system, the air-specific nature of the polypeptides is consistent with some potential involvement in the system. Since the polypeptides are all associated
136
SPALDING AND JEFFREY .:
200
b4
6
Plant Physiol. Vol. 89,1989
1
2 3 4 .-
6
_-s
E 8!
I*- 11967 --
97 68
~#-68
: ja tt t@;;'
_w
53
36
db 29C. *ad
18
X F4
Figure 3. Fluorograph of SDS-PAGE gel of low-speed membrane fractions from C02-ennched (lane 2), air-adapted (lane 7), and C02enriched cells transferred to limiting C02 for 1 h (lane 3), 2 h (lane 4), 4 h (lane 5), and 8 h (lane 6). Lanes 1 and 8 contain molecular size markers. Arrowheads indicate polypeptides apparently induced by limiting C02.
with membranes, it is additionally possible that one or more of them might be involved in inorganic carbon transport. It is also not clear with which membrane(s) the polypeptides are associated, although it seems clear they are not specifically associated with the thylakoid membranes. Crude membrane fractions were purposefully used in this study, because there is some controversy over the location of the inorganic carbon transport in eukaryotic microalgae (16). Following purification of the proteins and antibody production, we hope to be able to establish unambiguously the intracellular location using immunohistochemical methods. Coleman et al. (7) have previously demonstrated the specific occurrence of a 37 kD, soluble, periplasmic carbonic anhydrase in air-adapted versus CO,-enriched Chlamydomonas cells, and Coleman and Grossman (6) have identified a 20 kD soluble peptide specifically labeled with 35SO2-2 during adaptation of C02-enriched cells to limiting CO,. In addition, Badour and Kim (2) have reported air-specific polypeptides of 42, 22, and 20 kD in detergent extracts of whole cells of Chlamydomonas segnis. None of these polypeptides has been demonstrated to have any association with membranes but might be involved in the C02-concentrating system in some way. A soluble, 20 kD air-specific polypeptide was reported by Coleman and Grossman (6). We observed no obvious airspecific polypeptides of any size in our soluble fraction by either protein staining or "sS-labeling, but did occasionally observe a 21 kD soluble polypeptide by both methods to be faintly but specifically present in the soluble fraction of airadapted cells (not shown). We cannot currently explain this
Figure 4. Silver-stained SDS-PAGE gel of low-speed membrane fractions from C02-enriched (lane 2), air-adapted (lane 7), and C02enriched Ch. reinhardtii cells transferred to limiting C02 for 1 h (lane 3), 2 h (lane 4), 4 h (lane 5), and 8 h (lane 6). Lanes 1 and 8 contain molecular size markers. Arrowheads indicate polypeptides apparently induced by limiting C02.
-
El .2 c
SEs~~~~~~~~~~~~~~~~~~~~~~~ 4
c
UaE8
li 2
6
8
10
12
14
Hours After Transfer
Figure 5. Time course of increase in photosynthetic 14C02 fixation at limiting C02 (0) and intemal inorganic carbon accumulation (0) following transfer of C02-enriched Ch. reinhardtii to limiting C02. Assays for both parameters were performed simultaneously 30 s after introduction of 14CO2 (60 AM NaHCO3, pH 7.0).
apparent discrepancy with the work of Coleman and Grossman, but our soluble fraction was a 40,000g supernatant while theirs was a 1 35,000g supernatant. It is possible that the 20 kD polypeptide would be enriched, and therefore more apparent, in a 135,000g supematant. Since we were specifically interested in membrane-associated polypeptides, this was not investigated further. Coleman and Grossman (6) also reported two polypeptides
LIMITING-CO2 INDUCED POLYPEPTIDES IN CHLAMYDOMONAS
between 45 and 50 kD which were heavily 35S-labeled early in a time course of adaptation of C02-enriched Chlamydomonas cells to limiting C02. They observed a simultaneous decrease in the relative labeling of the Rubisco large and small subunits. The results described in this paper confirm those observations (Fig. 2). Although it cannot be ruled out at this point, it seems unlikely that either the 35 or 36 kD polypeptide described in this paper corresponds to the 37 kD periplasmic carbonic anhydrase described by Coleman et al. (7). In the cell-wallless Ch. reinhardtii strain (CW- 15) used here the periplasmic carbonic anhydrase is released into the growth medium (7) so would probably not be present in the cell extracts described in this paper. In the work described here, no evidence of a 37 kD air-specific polypeptide was seen by either protein staining or 35S-labeling in either the whole-cell or soluble protein fractions. It is still possible that the 36 or 35 kD (or both) polypeptide reported here represents a membrane-associated form of the periplasmic carbonic anhydrase. It is not clear whether any of the air-specific polypeptides reported by Badour and Kim (2) correspond to any of those reported in this paper. Since Badour and Kim extracted whole cells with a detergent mixture (2% Nonidet P40 and 0.04% SDS), it was not established whether the polypeptides reported by them were soluble or membrane-associated. Badour and Kim identified the air-specific polypeptides following twodimensional gel electrophoresis (IEF and SDS-PAGE). Because of the difficulties inherent in IEF ofmembrane proteins, it is not clear that membrane proteins would have been readily observed in their protocol. A large smear of protein was apparent on the alkaline edge of their two-dimensional gels which may represent many of the membrane proteins. It is possible, however, that the 22 and 20 kD air-specific polypeptides reported by Badour and Kim (2) correspond to the 21 and 19 kD air-specific polypeptides described in this paper. In addition to demonstrating four membrane-associated, air-specific polypeptides, a comparison of the kinetics of appearance of the four polypeptides with the appearance of the functional activity of the C02-concentrating system revealed that two of the polypeptides (Mr 21 and 36 kD) appeared with roughly the same kinetics as the C02-concentrating system. This correlation is consistent with a possible role in the C02-concentrating system, though it does not prove any involvement. The other two air-specific polypeptides (Mr 19 and 35 kD) were somewhat variable in their appearance and had a delayed appearance relative to the increase in the activity of the C02-concentrating system, so would appear to be less likely to be directly involved in the system. However, some involvement of these two polypeptides cannot be ruled out. In conclusion, four, membrane-associated polypeptides have been demonstrated to be specifically present in airadapted but not C02-enriched Chlamydomonas cells. The appearance of two of these polypeptides (Mr 21 and 36 kD) during induction of the C02-concentrating system correlate well with increase in the activity of the system. Although the results reported here are consistent with the possibility that one or more of these membrane-associated, air-specific polypeptides might be directly or indirectly involved in the mi-
137
croalgal C02-concentrating system, no involvement has actually been demonstrated. LITERATURE CITED 1. Badger MR, Kaplan A, Berry JA(1980) Internal inorganic carbon
2.
3. 4. 5.
6. 7.
8. 9. 10. 11.
12. 13. 14.
15.
16. 17.
18. 19. 20. 21.
22.
pool of Chlamydomonas reinhardtii. Evidence for a carbon dioxide concentrating mechanism. Plant Physiol 66: 407-413 Badour SS, Kim WK (1986) Comparison of polypeptides from Chiamydomonas segnis adapted to low and high carbon dioxide tensions. Biochem Physiol Pflanzen 181: 9-16 Beardall J (1981) CO2 accumulation by Chlorella saccharophila (Chlorophyceae) at low external pH: evidence for the active transport of inorganic carbon at the chloroplast envelope. J Phycol 17: 371-373 Beardall J, Raven JA (1981) Transport of inorganic carbon and the CO2 concentrating mechanism in Chlorella emersonii (Chlorophyceae). J Phycol 17: 134-141 Berry J, Boynton J, Kaplan A, Badger MR (1976) Growth and photosynthesis of Chlamydomonas reinhardtii as function of CO2 concentration. Carnegie Inst Wash Year Book 75: 423432 Coleman JR, Grossman AR (1983) Regulation of protein synthesis during adaptation of Chlamydomonas reinhardtii to low CO2- Carnegie Inst Wash Year Book 82: 109-111 Coleman JR, Berry JA, Togasaki RT, Grossman AR (1984) Identification of extracellular carbonic anhydrase of Chlamydomonas reinhardtii. Plant Physiol 76: 472-477 Greer KL, Plumley FG, Schmidt GW (1986) The water oxidation complex of Chlamydomonas. Accumulation and maturation of the largest subunit in photosystem II mutants. Plant Physiol 82: 114-120 Heukeshoven J, Dernick R (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6: 103-112 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680685 Marcus Y, Volokita M, Kaplan A (1984) The location of the transporting system for inorganic carbon and the nature of the form translocated in Chlamydomonas reinhardtii. J Exp Bot 35: 1136-1144 Moroney JV, Husic HD, Tolbert NE (1985) Effect of carbonic anhydrase inhibitors on inorganic carbon accumulation of Chlamydomonas reinhardtii. Plant Physiol 79: 177-183 Moroney JV, Kitayama M, Togasaki RK, Tolbert NE (1987) Evidence for inorganic carbon transport by intact chloroplasts of Chlamydomonas reinhardtii. Plant Physiol 83: 460-463 Piccioni R, Bellemare G, Chua N-H (1982) Methods ofpolyacrylamide gel electrophoresis in the analysis and preparation of plant polypeptides. In M Edelman, RB Hallick, N-H Chua, eds, Methods in Chloroplast Molecular Biology. Elsevier, Amsterdam, pp 985-1014 Skinner MK, Griswold MD (1983) Fluorographic detection of radioactivity in polyacrylamide gels with 2,5-diphenyloxazole in acetic acid and its comparison with existing procedures. Biochem J 209: 281-284 Spalding MH (1988) Photosynthesis and photorespiration in freshwater green algae. Aquat Bot (in press) Spalding MH, Spreitzer RJ, Ogren WL (1983) Carbonic anhydrase deficient mutant of Chlamydomonas reinhardii requires elevated carbon dioxide concentration for photoautotrophic growth. Plant Physiol 73: 268-272 Spalding MH, Spreitzer RJ, Ogren WL (1983) Reduced inorganic carbon transport in a C02-requiring mutant of Chlamydomonas reinhardii. Plant Physiol 73: 273-276 Spalding MH, Ogren WL (1983) Evidence for a saturable transport component in the inorganic carbon uptake of Chlamydomonas reinhardii. FEBS Lett 154: 335-338 Tsuzuki M, Miyachi S (1979) Effects of CO2 concentration during growth and of ethoxyzolamide on CO2 compensation point in Chlorella. FEBS Lett 103: 221-223 Winder T, Spalding MH (1988) Imazaquin and chlorsulfuron resistance in mutants of Chlamydomonas reinhardtii. Mol Gen Genet 213: 394-399 Zenvirth D, Kaplan A (1981) Uptake and efflux of inorganic carbon in Dunaliella salina. Planta 152: 8-12
