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Increased Turnover of a Polypeptide Associated with the Lightharvesting Chlorophyll-protein Complex II in Partially Bleached Euglena gracilis
J.PlantPhysiol. Vol. 136.pp. 187-192(1990)
Increased Turnover of a Polypeptide Associated with the Lightharvesting Chlorophyll-protein Complex II in Partially Bleached Euglena gracilis WILLIAM ORTIZ Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, Oklahoma 73019, USA Received May 16,1989 . Accepted December 7,1989
Summary The levels of a 24,500 Mr polypeptide of the light-harvesting chlorophyll-protein complex associated with photosystem II decrease in plastids of growing photo heterotrophic cultures of Euglena gracilis during heat-bleaching at 33°C. The polypeptide, however, continues to be synthesized, imported and incorporated into thylakoids despite reduced levels of accumulation. The failure of bleaching plastids to maintain normal levels of this polypeptide could be explained by an increase in turnover of the newly synthesized polypeptide at the elevated temperature. In this report, I show that the polypeptide is rapidly degraded when isolated plastids from [35S]labelled partially bleached Euglena are incubated at 33°C. Pulse-chase experiments carried out in vivo with partially bleached cell cultures essentially confirm the results obtained with isolated plastids. The results presented suggest that post-translational events regulate the levels of the 24,500 Mr polypeptide in bleaching Euglena.
Key words: Euglena gracilis Z; heat·bleaching; light-harvesting chlorophyll-protein complex II; post-translational regulation; protein turnover.
=
Abbreviations: ChI = chlorophyll; LHCPII = light-harvesting chlorophyll-protein complex II; PMSF phenyl methyl sulfonylfluoride.
Introduction
Growing photo heterotrophic cultures of the unicellular alga Euglena gracilis display a time-dependent loss of total chlorophyll (ChI) per cell at the moderately high temperature of 33 to 34°C (Pringsheim and Pringsheim, 1952). Prolonged growth at the elevated temperature, however, results in the production of irreversibly bleached cultures of the alga with an impaired capacity to carry out photosynthesis. This phenomenon also known as heat-bleaching offers an attractive opportunity to unveil some of the rules that govern the maintenance of chloroplasts as well as the contribution of the nuclear and chloroplast genetic centers to the events that unfold in chloroplasts especially during the early phases of the bleaching response. © 1990 by Gustav FIScher Verlag, Stuttgart
One of the most significant changes that takes place in plastids of Euglena grown at the elevated temperature is in the polypeptide composition of the light-harvesting chlorophyll alb-binding protein complex associated with Photosystem II (LHCPII) (Ortiz and Wilson, 1988). The levels of one of the polypeptides of the complex, a 24,500 Mr species, decrease at the elevated temperature while a second polypeptide of the complex (28,000 Mr) remains at levels comparable to the unbleached control. Since high temperature andlor loss of ChI could explain the preferential loss of the 24,500 Mr species from bleaching thylakoids it was important to establish which factor plays a direct role in regulating the levels of this polypeptide at the elevated temperature. We have previously shown that treatments that induce ChI loss such as darkness or treatments with levulinic acid affect dif-
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WILLIAM ORTIZ
ferently the ability of plastids to accumulate the polypeptide at room temperature (Ortiz and Kutner, 1989). Treatments with levulinic acid resulted in the loss of ChI on a cell basis and a loss of the 24,500 Mr polypeptide, whereas treatments in the dark led to a sharp decrease in ChI accumulation but no significant loss in the levels of this polypeptide during the initial 15 h incubation in the dark. Furthermore, the loss of the 24,500 Mr species at 33°C could not be correlated with a preferential loss of ChI b from thylakoids since ChI a/ChI b ratios actually decline during bleaching. Since ChI loss does not always result in a decline in the levels of the 24,500 Mr species, we suggested that temperature is perhaps the overriding factor affecting the ability of bleaching plastids to maintain this polypeptide at levels comparable to the control. Pulse-labelling experiments in vivo have shown that despite the reduced levels of this polypeptide, the 24,500 Mr species continues to be synthesized, imported and incorporated into the thylakoids of partially bleached plastids (Ortiz and Kutner, 1989). The failure of bleaching plastids to accumulate and maintain this polypeptide could be explained by a more rapid degradation of the newly synthesized polypeptide at the elevated temperature. In this report I compare the stability of the 24,500 Mr polypeptide in isolated plastids prepared from pulse-labelled unbleached and partially bleached (41 h) Euglena. The loss of the radiolabelled 24,500 Mr species in isolated plastids from 41 h bleached cells is faster at 33°C than at 23 0c. The same polypeptide, however, is quite stable in isolated chloroplasts (from unbleached cells) incubated at 33°C. The observations made with isolated plastids were confirmed with pulse-chase experiments on room temperature-grown and partially bleached (41 h) cells. The data presented suggest that the levels of the 24,500 Mr polypeptide in bleaching plastids is regulated at the post-translational level.
Materials and Methods Cell Culture Euglena gracilis Z. was grown photoheterotrophically at 23°C (unbleached control) or at 33 °C in modified Hutner's medium containing 50ng/L of vitamin BI2 (Ortiz et aI., 1980; Ortiz and Stutz, 1980). Cells were illuminated with cool-white fluorescent lamps at approximately 140ft-c and cultures were continuously shaken at 150 rpm. Pulse·labelling in vivo Algal cultures were grown at 23°C or at 33 °C for 41 h. Cells were harvested by centrifugation at 2,000 rpm for 1 min (Beckman JA-20 rotor) and resuspended in fresh low-sulfate heterotrophic medium in which MgS04 was replaced by MgCh (Monroy et aI., 1987). Radiolabelling in vivo was initiated by the addition of 8 /LCi of [35 S]sodium sulfate (43 Ci/mgS; ICN Biochemicals, Inc.) per ml of cell suspension followed by incubation at the original growth temperature in the light with continuous shaking for an additional 30 min.
Plastid isolation Radiolabelled cells were harvested by centrifugation and the isolated on isoosmotic linear gradients of Percoll (10 to
[3 5S]plastids
50 %) underlaid with a cushion of 80 % isoosmotic Percoll (Ortiz et aI., 1980; Ortiz and Wilson, 1988). The band of plastids was harvested from the gradient and washed by dilution with four volumes of ice cold gradient buffer followed by centrifugation at 5,000 rpm for 3 min (Beckman JA-20 rotor). The pellet of plastids was resuspended in ice cold gradient buffer to a concentration of approximately 250/Lg Chllml and kept in ice. Total Chi was determined according to Arnon (1949).
Temperature treatment 0/ isolated plastids and electrophoresis conditions The suspension of radiolabelled plastids from unbleached or 41 hbleached cells was incubated in the dark at 23°C or at 33 °C for 1 h. At selected time intervals 100/LL of suspension were withdrawn and immediately solubilized in electrophoresis sample buffer (Chua, 1980) containing 2M urea and 1 mM PMSF. Samples were loaded on an SDS-polyacrylamide 10 to 15 % linear gradient gel. Electrophoresis was carried out overnight at 6 mAmp constant current. Radiolabelled polypeptides on the gel were visualized by fluorography at -70°C (Laskey and Mills, 1975; Burckhardt et al., 1979).
Pulse-chase experiment and immunoprecipitation Cells grown at room temperature or at 33°C were harvested by centrifugation and resuspended in fresh low-sulfate heterotrophic medium (40mL) in which MgS04 was replaced by MgCh (Monroy et aI., 1987) at a cell concentration of 5 x 106 cells/mL. Cells were pulse-labelled at the original growth temperature in the light with 8/LCi of [35 S]sodium sulfate (43 Ci/mgS; ICN Biochemicals Inc.) per mL of cell suspension for an additional 2 h. At the end of the radiolabelling period (pulse) the cell suspension was divided into two flasks and the chase was initiated by the addition of nonradioactive Na2S04 to a final concentration of 10 mM. One flask was incubated at room temperature while the second flask was incubated at 33°C. Samples (300/LL) were taken at different intervals during the chase period and the LHCPII was immunoprecipitated according to the method of Merchant and Bogorad (1986) with antibody prepared in rabbits against the Chlamydomonas LHCPII. The antibody was a generous gift of Drs. G. W. Schmidt and F. G. Plumley, University of Georgia. The antigen-antibody complex was released from Protein ASepharose 6MB by solubilization in electrophoresis sample buffer (Chua, 1980) containing 2M urea and 1 mM PMSF. Samples were loaded on 2 M urea-SDS-polyacrylamide (12.5 %) gels and ran overnight at 6 mamp. Gels were prepared for fluorography according to Laskey and Mills (1975) with a modification introduced by Burckhardt et al. (1979). The radioactivity associated with the 24,500 Mr polypeptide was quantified by determining the area of the peaks on densitometric scans of the bands on the X-ray film.
Results Differences in the level of accumulation of the 24,500 Mr polypeptide of the LHCPII between unbleached and partially bleached Euglena could be attributed to a more rapid degradation of the newly synthesized polypeptide following incorporation into the thylakoids of bleaching Euglena. The fate of the newly synthesized/integrated polypeptide was studied first using isolated plastids from pulse-labelled unbleached and partially bleached Euglena incubated at 23 or 33°C in the dark for 1 h. Dark conditions were chosen in
Turnover of the 24,500 Mr polypeptide of the LHCPll
A
33°-33-C 33°--33°C 00 2040 60 2040 60
23-C 23°C 3060 3060
----- - -.. .... ------ -.........-...-. -
6060-
-..
--
lit ....
rr..
24.5
B
88
the newly synthesized/integrated polypeptide is rapidly degraded at the elevated temperature. By contrast, the radioactivity associated with other polypeptides (Fig. 1 A) does not decrease as rapidly (13,000 Mr) or shows little or no change (60,000 Mr). In fact, the amount of labelling associated with a majority of polypeptides on the fluorograph does not change appreciably at the elevated temperature. Although a rapid decline in the amount of radioactivity in the 24,500 Mr species is observed at 33°C, little change is observed in the radioactivity associated with this polypeptide at 23°C (Fig. 1 A, right). These results suggest that the decrease in radioactivity observed in the 24,500 Mr species is the result of a selective degradation of this polypeptide. Furthermore, the activity responsible for the degradation of the newly synthesized/integrated 24,500 Mr polypeptide appears to be sensitive to temperature since the same polypeptide in the same membrane environment (i.e., a partially bleached thylakoid) is not degraded at 23°C for at least 1 h. h. I sought to determine whether the 24,500 Mr polypeptide in isolated chloroplasts from room temperature grown cultures is also subjected to rapid degradation when isolated chloroplasts are incubated at 33°C. Cultures of control cells were pulse-labelled with [3sS]sodium sulfate, chloroplasts were isolated on isoosmotic gradients of Percoll and in-
23°-33°C o 20 40 60
15 15 20 20 40 40 60 60 0o
24.5-111 '.
24.5-
•
Fig. 1: Selective degradation of the 24,500 Mr polypeptide in isolated partially bleached (41 h) Euglena plastids. Cells were grown photoheterotrophically for 41 h at 33° and pulse-labelled with [35 S]SO_ dium sulfate for 30 min at the same temperature. Plastids were isolated and reincubated at 33°C (or at 23°C) for up to 60 min. At selected times, a 100 III aliquot was withdrawn and immediately mixed with sample buffer containing a final concentration of 2 M urea and 1 mM PMSF, followed by SDS-polyacrylamide gel electrophoresis and fluorography for the detection of radiolabelled polypeptides. (A) Fluorograph showing the complete array of radiolabelled plastid polypeptides. Each lane is identified by the time (in min) at which the sampling occurred. The solid arrowheads indicate the position of the protein standards: bovine serum albumin, ovalbumin, trypsinogen and lysozyme. The pol ypeptides identified by a number (Mr in thousands) are mentioned in the text. (B) A slice of a fluorograph showing the region corresponding to the 24,500 Mr polypeptide from a second independent experiment in which isolated partially bleached plastids were incubated at 33°C.
order to avoid possible photoxidative damage of membrane proteins in the light (Holloway and Dalling, 1988). Cultures of 41 h-bleached Euglena were pulsed with [3sS]sodium sulfate for 30 min in the light and plastids isolated on gradients of isoosmotic Percoll followed by incubation at 23°C or at 33 0c. In this experiment, the radioactivity present in the 24,500 Mr polypeptide decreases during a 1 h incubation period at 33°C (Figs. 1 A and 1 B) indicating that
189
.........
--lIllIlIil l
-
5454
...... 1· .........
43-3 " : - - - 4 24.524.5'
.111111
-Fig. 2: Stability of the 24,500 Mr polypeptide in isolated chloroplast from unbleached Euglena. Cultures of the alga were grown photoheterotrophically at 23°C and pulse-labelled with eSS]sodium sulfate for 30 min at the same temperature. The isolated chloroplasts were incubated at 33°C for up to 60 min. At selected intervals, a 100 III aliquot was sampled and immediately mixed with sample buffer containing a final concentration of 2 M urea and 1 mM PMSF. Polypeptides were fractionated by SDS-polyacrylamide gel electrophoresis followed by fluorography. The number above each lane indicates the time of sampling in min . Solid arrowheads indicate the position of the standards identified in Fig. 1. The polypeptides identified by a number (Mr in thousands) are specifically mentioned in the text.
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WILUAM ORTIZ ORTIZ WILLIAM
33°-33°C 20 40 60 90
.. _.,. . ....... o
.....- ... oo
20 40 40 60 60 90 90 20
'.
Fig. 3: Rapid degradation of the newly synthesized 24,500 Mr polypeptide of the LHCPII in partially bleached Euglena. Cultures of 41 h-bleached photoheterotrophic Euglena were pulse-labelled with [JSS]sodium sulfate for 2 h at 33°C. The chase period was initiated by the addition of nonradioactive sodium sulfate (10 mM final concentration). Half the culture was chased at 33°C (upper panel) while the other half was chased at 23 °C (lower panel). Cells were sampled at the times indicated (in min) and solubilized with detergent. Immunoprecipitation was carried out with antibody to the LHCPII of Chlamydomonas reinhardi. The immunoprecipitate was separated by urea-SDS-polyacrylamide gel electrophoresis followed by flu orography, fluorography.
cubated at 33°C for 1 h. No apparent degradation of the 24,500Mr polypeptide or of any of the other radiolabelled polypeptides on the fluorograph (such as 54,000 and a 43,000 Mr species) is observed when isolated chloroplasts from room temperature grown cultures are incubated at (Fig.2) 33°C (Fig. 2) or at 23°C (data not shown). The results suggest that the newly synthesized/integrated 24,500 Mr polypeptide in control cultures of Euglena is stable at both temperatures at least during short term incubations of 1 h. Although a rapid degradation of the 24,500 Mr polypeptide could be demonstrated in isolated plastids from partially bleached (41 h) cultures, pulse-chase experiments were also carried out to confirm the observations made with isolated plastids. In the first experiment cultures of Euglena were grown at 33°C for 41 h. The partially bleached cells were pulse-labelled with [3S]sodium sulfate for another 2 h at 33°C followed by the addition of nonradioactive sodium sulfate (final concentration of 10 mM) to mark the beginning of a 90 min chase period. Half the culture was chased at 23°C while the remaining half was incubated at 33 dc. 0c. Aliquots were withdrawn at different intervals and the LHCPII was Was immunoprecipitated from the detergent solubilized cells using antibody to to the Chlamydomonas LHCPII. The immunoprecipitated material was separated by electrophoresis followed by fluorography (Fig. (Fig. 3).
The upper panel shows the results from the experiment in which the partially bleached culture was pulsed and chased at 33°C. A rapid degradation of the 24,500 Mr polypeptide is evident and only traces of radioactivity are detected at 40, 60 and 90 min following the onset of the chase period. The amount of radioactivity associated with the immunoprecipitated polypeptide was quantified from the intensity of the band on the fluorograph. A semilogarithmic plot of this data (not shown) resulted in an estimated half-life for this polypeptide of 25 min in partially bleached (41 h) cultures. This half-life compares favorably with the estimated 35 min halflife from the experiments involving isolated plastids illustrated in Fig. 1. The lower panel in Fig. 3 shows a much slower degradation of the 24,500 Mr species in vivo when pulse-labelled partially bleached cultures were chased with unlabelled sulfate at 23°C. Although loss of radioactivity is slower at 23 °C than at 33°C, the degradation of the 24,500 Mr polypeptide in vivo is nearly complete after 90 min at 23°C. The result from the in vivo chase experiment carried out at 23°C 23 °C appears to be somewhat at odds with the earlier in vitro experiment (Fig. 1) in which isolated partially bleached plastids were incubated at 23°C. Although no significant loss of radioactivity was reported for the 24,500 Mr species in the in vitro experiment, the shorter total incubation period (60 min) in the experiment could explain why no significant degradation of the 24,500 Mr was detected when isolated plastids were incubated at 23°C. In this regard, some loss of radioactivity could be detected in the 24,500 Mr species after 60 min in the in vivo chase experiment carried out at 23°C (Fig. 3). Degradation of the polypeptide, however, is nearly complete after 90 min since only traces of radioactivity could be detected at this time. Based on the results from the in vivo pulse-chase experiment in Fig. 3 which involved
min
_ 33° -----33° 23° 120 120
Fig. 4: Stability of the newly synthesized 24,500 Mr polypeptide in Euglena. Cultures of photoheterotrophic room temperature grown Euglena. Euglena grown at room temperature were pulse-labelled with 35 S]sodium sulfate at 23°C for 2 h. The chase period was initiated [[3S by adding non-radioactive sodium sulfate (10 mM final concentration). Half the culture was chased for 2 h at 33°C while the other 0c. Cells were sampled at the time indicated half was chased at 23 dc. (in min) and solubilized with detergent. Immunoprecipitation was carried out with antibody to the Chlamydomonas LHCPII. The imby electrophoresis on urea-SDSmunoprecipitate was separated by f1uorography. polyacrylamide gels followed by fluorography.
Turnover of the 24,500 Mr polypeptide of the LHCPII longer incubation times (90 min) it is possible to conclude that despite a slower degradation at 23°C, the 24,500 Mr species is not maintained in partially bleached cells incubated at the normal growth temperature. Pulse-chase experiments were also conducted with room temperature grown cultures of Euglena using the same basic strategy described earlier. The cells were pulse-labelled at 23°C and the chase period was carried out for 2 h at 23°C or at 33 °C (Fig. 4). The results of this experiment show that the 24,500 Mr polypeptide in control cultures is not rapidly degraded at 23°C or at 33 These observations support earlier conclusions from work involving isolated chloroplasts (Fig. 2). Altogether both experimental approaches support the notion that the 24,500 Mr polypeptide is stably maintained in control cells.
0c.
Discussion Factors that interfere with the normal development and maintenance of chloroplasts limit the ability of plant cells to carry out photosynthesis. In this regard, the phenomenon of heat-bleaching in Euglena gracilis is particularly relevant since bleaching ultimately leads to the irreversible loss of photosynthetic competence in growing cultures incubated at the moderately elevated temperature of 33°C. Bleaching in Euglena is, in our view, an attractive model system in which to study some of the rules that govern the development and maintenance of this organelle in plant cells. Growth at the elevated temperature impairs the ability of Euglena cells to maintain functional chloroplasts. This, we propose, is the result of changes in the flow of genetic information from the two genetic compartments, the nucleocytoplasm and the chloroplast, that contribute to the maintenance of chloroplast function in plant cells (Ellis, 1981). The loss of total ChI that accompanies growth of Euglena cultures at the bleaching temperature has prompted us to study the effect of temperature on the synthesis and accumulation of ChI-binding polypeptides in bleaching plastids. The polypeptides of the LHCPII are particularly attractive to us since they are one of the most abundant polypeptides in thylakoids. These polypeptides bind ChI a and ChI b noncovalently and organize the photosynthetic pigments in a way that ensures the efficient transfer of excitation energy to the reaction center. Genetic information for the polypeptides of the LHCPII resides in the nucleus (Tobin and Silverthorne, 1985). The messenger RNA is translated on cytoplasmic ribosomes and the product, a precursor polypeptide, is then imported by the chloroplast, processed and targeted to the thylakoid. In this regard, the effect of temperature on the ability of plastids to accumulate polypeptides of nucleocytoplasmic origin, such as the polypeptides of the LHCPII, could provide important insight on the molecular mechanisms that underlie the loss of photosynthetic competence in Euglena at the moderate temperature of 33°C. Although the levels of the 24,500 Mr polypeptide of the LHCPII decrease during temperature-induced bleaching in Euglena gracilis, the polypeptide continues to be synthesized, imported and incorporated into thylakoids of bleaching
191
plastids (Ortiz and Kutner, 1989). This observation prompted the suggestion that the levels of the polypeptide in bleaching plastids are controlled at the post-translational level through the rapid degradation of the polypeptide following its incorporation into the thylakoid. Degradation of the 24,500 Mr polypeptide in partially bleached (41 h) Euglena was demonstrated by two different experimental approaches involving isolated plastids and pulse-chase experiments using whole cells. Based on the results from the pulse-chase experiments it is estimated that the half-life of the 24,500 Mr species is 25 minutes in partially bleached cells. The same polypeptide, however, is maintained in control cultures at 33°C for at least 2 h. Furthermore, the activity responsible for the degradation of this polypeptide of the LHCPII appears to be selective since only the 24,500 Mr species is significantly degraded in isolated plastids from partially bleached cells during the 1 h incubation period at 33°C. Although the 24,500 Mr polypeptide is not degraded when unbleached cultures of Euglena are incubated at 33°C for at least 2 h, the 24,500 Mr species is degraded in partially bleached cells incubated at 23°C or at 33 °C (pulse-chase experiments). These observations suggest that the membrane environment in partially bleached thylakoids is different from the membrane environment in control thylakoids and does not contribute to the stable integration of the polypeptide into partially bleached thylakoids. The membrane environment in thylakoids from control cells, on the other hand, permits the stable integration and maintenance of the newly synthesized polypeptide for at least 2 h at 33°C. Events at the post-translational level have been implicated in the regulation of chloroplast polypeptides such as the large (Chen et aI., 1988) and small (Schmidt and Mishkind, 1983) subunit of ribulose 1,5-bisphosphate carboxylase, plastocyanin (Merchant and Bogorad, 1986), subunits of the chloroplast coupling factor (Biekmann and Feierabend, 1985), and the QB-binding protein (Mattoo et aI., 1984). The seemingly widespread occurrence of post-translational regulation suggests that events at this level of regulation play an important role in maintaining a precise stoichiometric balance of photosynthetic proteins especially those that are components of multimeric complexes and in the removal of damaged or dispensable proteins from plastids. Acknowledgements The author thanks Drs. G. W. Schmidt and F. G. Plumley for their generous gift of antibody to the LHCPII of Chlamydomonas reinhardii and Ms. Beverly Richey for typing this manuscript. This work was supported by a grant from the National Science Foundation (DCB-8715422).
References ARNON, D. I.: Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). BIEKMANN, S. and J. FEIERABEND: Synthesis and degradation of unassembled polypeptides of the coupling factor of photophosphorylation CF 1 in 70S ribosome-deficient rye leaves. Eur. J. Biochem. 152, 529-535 (1985).
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BURCKHARDT, J., J. TELFORD, and M. L. BIRNSTIEL: Detection of labelled RNA species by contact hybridization. Nucleic Acid Res. 6,2963-2971 (1979). CHEN, Z., J. CHASTAIN, S. R. AL-ABED, R. CHOLLET, and R. J. SPREITZER: Reduced CO 2/0 2 specificity of ribulose-bisphosphate carboxylase/oxygenase in a temperature-sensitive chloroplast mutant of Chlamydomonas. Proc. Nat. Acad. Sci. USA 85, 4696-4699 (1988). CHUA, N. H.: Electrophoretic analysis of chloroplast proteins. Methods in Enzymo!. 69, 434-446 (1980). Ems, R. J.: Chloroplast proteins: synthesis, transport and assembly. Ann. Rev. Plant Physio!. 32, 111-137 (1981). HOLLOWAY, P. J. and M. J. DALLING: Modulation of the light-stimulated loss of polypeptides from isolated wheat thylakoids in vitro. J. Plant Physio!. 132,273-278 (1988). LASKEY, R. A. and D. MILLS: Quantitative film detection of 3H and J4C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341 (1975). MArroo, A. K., H. HOFFMAN-FALK, J. B. MARDER, and M. EDELMAN: Regulation of protein metabolism: coupling of photosynthetic electron transport to in vivo degradation of the rapidly metabolized 32-kilodalton protein of the chloroplast membranes. Proc. Nat. Acad. Sci. USA 81, 1380-1384 (1984). MERCHANT, S. and L. BOGORAD: Rapid degradation of apoplastocyanin in Cu(II)-deficient cells of Chlamydomonas reinhardtii. J. Bio!. Chern. 261, 15850-15853 (1986).
c.
MONROY, A. F., S. A. MCCARTHY, and S. D. SCHWARTZBACH: Evidence for translational regulation of chloroplast and mitochondrial biogenesis in Euglena. Plant Science 51, 61-76 (1987). ORTIZ, W. and M. S. KUTNER: Factors that affect the synthesis and accumulation of a polypeptide of the light-harvesting chlorophyll-protein complex II in Euglena gractlis. J. Plant Physiol. 134, 75-80 (1989). ORTIZ, W., E. M. REARDON, and C. A. PRICE: Preparation of chloroplasts from Euglena highly active in protein synthesis. Plant Physio!' 66, 291- 294 (1980). ORTIZ, W . and E. STUTZ: Synthesis of polypeptides of the chlorophyll-protein complexes in isolated chloroplasts of Euglena gra· cilis. FEBS Lett. 116, 298 - 302 (1980). ORTIZ, W. and C. J. WILSON: Induced changes in chloroplast protein accumulation during heat bleaching in Euglena gracilis. Plant Physio!. 86,554-561 (1988). PRINGSHEIM, E. G. and O. PRINGSHEIM: Experimental elimination of chromatophores and eye-spot in Euglena gractlts. New Phytol. 51,65-76 (1952). SCHMIDT, G. W. and M. L. MISHKIND: Rapid degradation of unassembled ribulose 1,5-bisphosphate carboxylase small subunits in chloroplasts. Proc. Nat. Acad. Sci. USA 80, 2632-2636 (1983). TOBIN, E. M. and J. SILVERTHORNE: Light regulation of gene expression in higher plants. Ann. Rev. Plant Physio!. 36, 569-594 (1985).
Increased Turnover of a Polypeptide Associated with the Lightharvesting Chlorophyll-protein Complex II in Partially Bleached Euglena gracilis WILLIAM ORTIZ Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, Oklahoma 73019, USA Received May 16,1989 . Accepted December 7,1989
Summary The levels of a 24,500 Mr polypeptide of the light-harvesting chlorophyll-protein complex associated with photosystem II decrease in plastids of growing photo heterotrophic cultures of Euglena gracilis during heat-bleaching at 33°C. The polypeptide, however, continues to be synthesized, imported and incorporated into thylakoids despite reduced levels of accumulation. The failure of bleaching plastids to maintain normal levels of this polypeptide could be explained by an increase in turnover of the newly synthesized polypeptide at the elevated temperature. In this report, I show that the polypeptide is rapidly degraded when isolated plastids from [35S]labelled partially bleached Euglena are incubated at 33°C. Pulse-chase experiments carried out in vivo with partially bleached cell cultures essentially confirm the results obtained with isolated plastids. The results presented suggest that post-translational events regulate the levels of the 24,500 Mr polypeptide in bleaching Euglena.
Key words: Euglena gracilis Z; heat·bleaching; light-harvesting chlorophyll-protein complex II; post-translational regulation; protein turnover.
=
Abbreviations: ChI = chlorophyll; LHCPII = light-harvesting chlorophyll-protein complex II; PMSF phenyl methyl sulfonylfluoride.
Introduction
Growing photo heterotrophic cultures of the unicellular alga Euglena gracilis display a time-dependent loss of total chlorophyll (ChI) per cell at the moderately high temperature of 33 to 34°C (Pringsheim and Pringsheim, 1952). Prolonged growth at the elevated temperature, however, results in the production of irreversibly bleached cultures of the alga with an impaired capacity to carry out photosynthesis. This phenomenon also known as heat-bleaching offers an attractive opportunity to unveil some of the rules that govern the maintenance of chloroplasts as well as the contribution of the nuclear and chloroplast genetic centers to the events that unfold in chloroplasts especially during the early phases of the bleaching response. © 1990 by Gustav FIScher Verlag, Stuttgart
One of the most significant changes that takes place in plastids of Euglena grown at the elevated temperature is in the polypeptide composition of the light-harvesting chlorophyll alb-binding protein complex associated with Photosystem II (LHCPII) (Ortiz and Wilson, 1988). The levels of one of the polypeptides of the complex, a 24,500 Mr species, decrease at the elevated temperature while a second polypeptide of the complex (28,000 Mr) remains at levels comparable to the unbleached control. Since high temperature andlor loss of ChI could explain the preferential loss of the 24,500 Mr species from bleaching thylakoids it was important to establish which factor plays a direct role in regulating the levels of this polypeptide at the elevated temperature. We have previously shown that treatments that induce ChI loss such as darkness or treatments with levulinic acid affect dif-
188
WILLIAM ORTIZ
ferently the ability of plastids to accumulate the polypeptide at room temperature (Ortiz and Kutner, 1989). Treatments with levulinic acid resulted in the loss of ChI on a cell basis and a loss of the 24,500 Mr polypeptide, whereas treatments in the dark led to a sharp decrease in ChI accumulation but no significant loss in the levels of this polypeptide during the initial 15 h incubation in the dark. Furthermore, the loss of the 24,500 Mr species at 33°C could not be correlated with a preferential loss of ChI b from thylakoids since ChI a/ChI b ratios actually decline during bleaching. Since ChI loss does not always result in a decline in the levels of the 24,500 Mr species, we suggested that temperature is perhaps the overriding factor affecting the ability of bleaching plastids to maintain this polypeptide at levels comparable to the control. Pulse-labelling experiments in vivo have shown that despite the reduced levels of this polypeptide, the 24,500 Mr species continues to be synthesized, imported and incorporated into the thylakoids of partially bleached plastids (Ortiz and Kutner, 1989). The failure of bleaching plastids to accumulate and maintain this polypeptide could be explained by a more rapid degradation of the newly synthesized polypeptide at the elevated temperature. In this report I compare the stability of the 24,500 Mr polypeptide in isolated plastids prepared from pulse-labelled unbleached and partially bleached (41 h) Euglena. The loss of the radiolabelled 24,500 Mr species in isolated plastids from 41 h bleached cells is faster at 33°C than at 23 0c. The same polypeptide, however, is quite stable in isolated chloroplasts (from unbleached cells) incubated at 33°C. The observations made with isolated plastids were confirmed with pulse-chase experiments on room temperature-grown and partially bleached (41 h) cells. The data presented suggest that the levels of the 24,500 Mr polypeptide in bleaching plastids is regulated at the post-translational level.
Materials and Methods Cell Culture Euglena gracilis Z. was grown photoheterotrophically at 23°C (unbleached control) or at 33 °C in modified Hutner's medium containing 50ng/L of vitamin BI2 (Ortiz et aI., 1980; Ortiz and Stutz, 1980). Cells were illuminated with cool-white fluorescent lamps at approximately 140ft-c and cultures were continuously shaken at 150 rpm. Pulse·labelling in vivo Algal cultures were grown at 23°C or at 33 °C for 41 h. Cells were harvested by centrifugation at 2,000 rpm for 1 min (Beckman JA-20 rotor) and resuspended in fresh low-sulfate heterotrophic medium in which MgS04 was replaced by MgCh (Monroy et aI., 1987). Radiolabelling in vivo was initiated by the addition of 8 /LCi of [35 S]sodium sulfate (43 Ci/mgS; ICN Biochemicals, Inc.) per ml of cell suspension followed by incubation at the original growth temperature in the light with continuous shaking for an additional 30 min.
Plastid isolation Radiolabelled cells were harvested by centrifugation and the isolated on isoosmotic linear gradients of Percoll (10 to
[3 5S]plastids
50 %) underlaid with a cushion of 80 % isoosmotic Percoll (Ortiz et aI., 1980; Ortiz and Wilson, 1988). The band of plastids was harvested from the gradient and washed by dilution with four volumes of ice cold gradient buffer followed by centrifugation at 5,000 rpm for 3 min (Beckman JA-20 rotor). The pellet of plastids was resuspended in ice cold gradient buffer to a concentration of approximately 250/Lg Chllml and kept in ice. Total Chi was determined according to Arnon (1949).
Temperature treatment 0/ isolated plastids and electrophoresis conditions The suspension of radiolabelled plastids from unbleached or 41 hbleached cells was incubated in the dark at 23°C or at 33 °C for 1 h. At selected time intervals 100/LL of suspension were withdrawn and immediately solubilized in electrophoresis sample buffer (Chua, 1980) containing 2M urea and 1 mM PMSF. Samples were loaded on an SDS-polyacrylamide 10 to 15 % linear gradient gel. Electrophoresis was carried out overnight at 6 mAmp constant current. Radiolabelled polypeptides on the gel were visualized by fluorography at -70°C (Laskey and Mills, 1975; Burckhardt et al., 1979).
Pulse-chase experiment and immunoprecipitation Cells grown at room temperature or at 33°C were harvested by centrifugation and resuspended in fresh low-sulfate heterotrophic medium (40mL) in which MgS04 was replaced by MgCh (Monroy et aI., 1987) at a cell concentration of 5 x 106 cells/mL. Cells were pulse-labelled at the original growth temperature in the light with 8/LCi of [35 S]sodium sulfate (43 Ci/mgS; ICN Biochemicals Inc.) per mL of cell suspension for an additional 2 h. At the end of the radiolabelling period (pulse) the cell suspension was divided into two flasks and the chase was initiated by the addition of nonradioactive Na2S04 to a final concentration of 10 mM. One flask was incubated at room temperature while the second flask was incubated at 33°C. Samples (300/LL) were taken at different intervals during the chase period and the LHCPII was immunoprecipitated according to the method of Merchant and Bogorad (1986) with antibody prepared in rabbits against the Chlamydomonas LHCPII. The antibody was a generous gift of Drs. G. W. Schmidt and F. G. Plumley, University of Georgia. The antigen-antibody complex was released from Protein ASepharose 6MB by solubilization in electrophoresis sample buffer (Chua, 1980) containing 2M urea and 1 mM PMSF. Samples were loaded on 2 M urea-SDS-polyacrylamide (12.5 %) gels and ran overnight at 6 mamp. Gels were prepared for fluorography according to Laskey and Mills (1975) with a modification introduced by Burckhardt et al. (1979). The radioactivity associated with the 24,500 Mr polypeptide was quantified by determining the area of the peaks on densitometric scans of the bands on the X-ray film.
Results Differences in the level of accumulation of the 24,500 Mr polypeptide of the LHCPII between unbleached and partially bleached Euglena could be attributed to a more rapid degradation of the newly synthesized polypeptide following incorporation into the thylakoids of bleaching Euglena. The fate of the newly synthesized/integrated polypeptide was studied first using isolated plastids from pulse-labelled unbleached and partially bleached Euglena incubated at 23 or 33°C in the dark for 1 h. Dark conditions were chosen in
Turnover of the 24,500 Mr polypeptide of the LHCPll
A
33°-33-C 33°--33°C 00 2040 60 2040 60
23-C 23°C 3060 3060
----- - -.. .... ------ -.........-...-. -
6060-
-..
--
lit ....
rr..
24.5
B
88
the newly synthesized/integrated polypeptide is rapidly degraded at the elevated temperature. By contrast, the radioactivity associated with other polypeptides (Fig. 1 A) does not decrease as rapidly (13,000 Mr) or shows little or no change (60,000 Mr). In fact, the amount of labelling associated with a majority of polypeptides on the fluorograph does not change appreciably at the elevated temperature. Although a rapid decline in the amount of radioactivity in the 24,500 Mr species is observed at 33°C, little change is observed in the radioactivity associated with this polypeptide at 23°C (Fig. 1 A, right). These results suggest that the decrease in radioactivity observed in the 24,500 Mr species is the result of a selective degradation of this polypeptide. Furthermore, the activity responsible for the degradation of the newly synthesized/integrated 24,500 Mr polypeptide appears to be sensitive to temperature since the same polypeptide in the same membrane environment (i.e., a partially bleached thylakoid) is not degraded at 23°C for at least 1 h. h. I sought to determine whether the 24,500 Mr polypeptide in isolated chloroplasts from room temperature grown cultures is also subjected to rapid degradation when isolated chloroplasts are incubated at 33°C. Cultures of control cells were pulse-labelled with [3sS]sodium sulfate, chloroplasts were isolated on isoosmotic gradients of Percoll and in-
23°-33°C o 20 40 60
15 15 20 20 40 40 60 60 0o
24.5-111 '.
24.5-
•
Fig. 1: Selective degradation of the 24,500 Mr polypeptide in isolated partially bleached (41 h) Euglena plastids. Cells were grown photoheterotrophically for 41 h at 33° and pulse-labelled with [35 S]SO_ dium sulfate for 30 min at the same temperature. Plastids were isolated and reincubated at 33°C (or at 23°C) for up to 60 min. At selected times, a 100 III aliquot was withdrawn and immediately mixed with sample buffer containing a final concentration of 2 M urea and 1 mM PMSF, followed by SDS-polyacrylamide gel electrophoresis and fluorography for the detection of radiolabelled polypeptides. (A) Fluorograph showing the complete array of radiolabelled plastid polypeptides. Each lane is identified by the time (in min) at which the sampling occurred. The solid arrowheads indicate the position of the protein standards: bovine serum albumin, ovalbumin, trypsinogen and lysozyme. The pol ypeptides identified by a number (Mr in thousands) are mentioned in the text. (B) A slice of a fluorograph showing the region corresponding to the 24,500 Mr polypeptide from a second independent experiment in which isolated partially bleached plastids were incubated at 33°C.
order to avoid possible photoxidative damage of membrane proteins in the light (Holloway and Dalling, 1988). Cultures of 41 h-bleached Euglena were pulsed with [3sS]sodium sulfate for 30 min in the light and plastids isolated on gradients of isoosmotic Percoll followed by incubation at 23°C or at 33 0c. In this experiment, the radioactivity present in the 24,500 Mr polypeptide decreases during a 1 h incubation period at 33°C (Figs. 1 A and 1 B) indicating that
189
.........
--lIllIlIil l
-
5454
...... 1· .........
43-3 " : - - - 4 24.524.5'
.111111
-Fig. 2: Stability of the 24,500 Mr polypeptide in isolated chloroplast from unbleached Euglena. Cultures of the alga were grown photoheterotrophically at 23°C and pulse-labelled with eSS]sodium sulfate for 30 min at the same temperature. The isolated chloroplasts were incubated at 33°C for up to 60 min. At selected intervals, a 100 III aliquot was sampled and immediately mixed with sample buffer containing a final concentration of 2 M urea and 1 mM PMSF. Polypeptides were fractionated by SDS-polyacrylamide gel electrophoresis followed by fluorography. The number above each lane indicates the time of sampling in min . Solid arrowheads indicate the position of the standards identified in Fig. 1. The polypeptides identified by a number (Mr in thousands) are specifically mentioned in the text.
190
WILUAM ORTIZ ORTIZ WILLIAM
33°-33°C 20 40 60 90
.. _.,. . ....... o
.....- ... oo
20 40 40 60 60 90 90 20
'.
Fig. 3: Rapid degradation of the newly synthesized 24,500 Mr polypeptide of the LHCPII in partially bleached Euglena. Cultures of 41 h-bleached photoheterotrophic Euglena were pulse-labelled with [JSS]sodium sulfate for 2 h at 33°C. The chase period was initiated by the addition of nonradioactive sodium sulfate (10 mM final concentration). Half the culture was chased at 33°C (upper panel) while the other half was chased at 23 °C (lower panel). Cells were sampled at the times indicated (in min) and solubilized with detergent. Immunoprecipitation was carried out with antibody to the LHCPII of Chlamydomonas reinhardi. The immunoprecipitate was separated by urea-SDS-polyacrylamide gel electrophoresis followed by flu orography, fluorography.
cubated at 33°C for 1 h. No apparent degradation of the 24,500Mr polypeptide or of any of the other radiolabelled polypeptides on the fluorograph (such as 54,000 and a 43,000 Mr species) is observed when isolated chloroplasts from room temperature grown cultures are incubated at (Fig.2) 33°C (Fig. 2) or at 23°C (data not shown). The results suggest that the newly synthesized/integrated 24,500 Mr polypeptide in control cultures of Euglena is stable at both temperatures at least during short term incubations of 1 h. Although a rapid degradation of the 24,500 Mr polypeptide could be demonstrated in isolated plastids from partially bleached (41 h) cultures, pulse-chase experiments were also carried out to confirm the observations made with isolated plastids. In the first experiment cultures of Euglena were grown at 33°C for 41 h. The partially bleached cells were pulse-labelled with [3S]sodium sulfate for another 2 h at 33°C followed by the addition of nonradioactive sodium sulfate (final concentration of 10 mM) to mark the beginning of a 90 min chase period. Half the culture was chased at 23°C while the remaining half was incubated at 33 dc. 0c. Aliquots were withdrawn at different intervals and the LHCPII was Was immunoprecipitated from the detergent solubilized cells using antibody to to the Chlamydomonas LHCPII. The immunoprecipitated material was separated by electrophoresis followed by fluorography (Fig. (Fig. 3).
The upper panel shows the results from the experiment in which the partially bleached culture was pulsed and chased at 33°C. A rapid degradation of the 24,500 Mr polypeptide is evident and only traces of radioactivity are detected at 40, 60 and 90 min following the onset of the chase period. The amount of radioactivity associated with the immunoprecipitated polypeptide was quantified from the intensity of the band on the fluorograph. A semilogarithmic plot of this data (not shown) resulted in an estimated half-life for this polypeptide of 25 min in partially bleached (41 h) cultures. This half-life compares favorably with the estimated 35 min halflife from the experiments involving isolated plastids illustrated in Fig. 1. The lower panel in Fig. 3 shows a much slower degradation of the 24,500 Mr species in vivo when pulse-labelled partially bleached cultures were chased with unlabelled sulfate at 23°C. Although loss of radioactivity is slower at 23 °C than at 33°C, the degradation of the 24,500 Mr polypeptide in vivo is nearly complete after 90 min at 23°C. The result from the in vivo chase experiment carried out at 23°C 23 °C appears to be somewhat at odds with the earlier in vitro experiment (Fig. 1) in which isolated partially bleached plastids were incubated at 23°C. Although no significant loss of radioactivity was reported for the 24,500 Mr species in the in vitro experiment, the shorter total incubation period (60 min) in the experiment could explain why no significant degradation of the 24,500 Mr was detected when isolated plastids were incubated at 23°C. In this regard, some loss of radioactivity could be detected in the 24,500 Mr species after 60 min in the in vivo chase experiment carried out at 23°C (Fig. 3). Degradation of the polypeptide, however, is nearly complete after 90 min since only traces of radioactivity could be detected at this time. Based on the results from the in vivo pulse-chase experiment in Fig. 3 which involved
min
_ 33° -----33° 23° 120 120
Fig. 4: Stability of the newly synthesized 24,500 Mr polypeptide in Euglena. Cultures of photoheterotrophic room temperature grown Euglena. Euglena grown at room temperature were pulse-labelled with 35 S]sodium sulfate at 23°C for 2 h. The chase period was initiated [[3S by adding non-radioactive sodium sulfate (10 mM final concentration). Half the culture was chased for 2 h at 33°C while the other 0c. Cells were sampled at the time indicated half was chased at 23 dc. (in min) and solubilized with detergent. Immunoprecipitation was carried out with antibody to the Chlamydomonas LHCPII. The imby electrophoresis on urea-SDSmunoprecipitate was separated by f1uorography. polyacrylamide gels followed by fluorography.
Turnover of the 24,500 Mr polypeptide of the LHCPII longer incubation times (90 min) it is possible to conclude that despite a slower degradation at 23°C, the 24,500 Mr species is not maintained in partially bleached cells incubated at the normal growth temperature. Pulse-chase experiments were also conducted with room temperature grown cultures of Euglena using the same basic strategy described earlier. The cells were pulse-labelled at 23°C and the chase period was carried out for 2 h at 23°C or at 33 °C (Fig. 4). The results of this experiment show that the 24,500 Mr polypeptide in control cultures is not rapidly degraded at 23°C or at 33 These observations support earlier conclusions from work involving isolated chloroplasts (Fig. 2). Altogether both experimental approaches support the notion that the 24,500 Mr polypeptide is stably maintained in control cells.
0c.
Discussion Factors that interfere with the normal development and maintenance of chloroplasts limit the ability of plant cells to carry out photosynthesis. In this regard, the phenomenon of heat-bleaching in Euglena gracilis is particularly relevant since bleaching ultimately leads to the irreversible loss of photosynthetic competence in growing cultures incubated at the moderately elevated temperature of 33°C. Bleaching in Euglena is, in our view, an attractive model system in which to study some of the rules that govern the development and maintenance of this organelle in plant cells. Growth at the elevated temperature impairs the ability of Euglena cells to maintain functional chloroplasts. This, we propose, is the result of changes in the flow of genetic information from the two genetic compartments, the nucleocytoplasm and the chloroplast, that contribute to the maintenance of chloroplast function in plant cells (Ellis, 1981). The loss of total ChI that accompanies growth of Euglena cultures at the bleaching temperature has prompted us to study the effect of temperature on the synthesis and accumulation of ChI-binding polypeptides in bleaching plastids. The polypeptides of the LHCPII are particularly attractive to us since they are one of the most abundant polypeptides in thylakoids. These polypeptides bind ChI a and ChI b noncovalently and organize the photosynthetic pigments in a way that ensures the efficient transfer of excitation energy to the reaction center. Genetic information for the polypeptides of the LHCPII resides in the nucleus (Tobin and Silverthorne, 1985). The messenger RNA is translated on cytoplasmic ribosomes and the product, a precursor polypeptide, is then imported by the chloroplast, processed and targeted to the thylakoid. In this regard, the effect of temperature on the ability of plastids to accumulate polypeptides of nucleocytoplasmic origin, such as the polypeptides of the LHCPII, could provide important insight on the molecular mechanisms that underlie the loss of photosynthetic competence in Euglena at the moderate temperature of 33°C. Although the levels of the 24,500 Mr polypeptide of the LHCPII decrease during temperature-induced bleaching in Euglena gracilis, the polypeptide continues to be synthesized, imported and incorporated into thylakoids of bleaching
191
plastids (Ortiz and Kutner, 1989). This observation prompted the suggestion that the levels of the polypeptide in bleaching plastids are controlled at the post-translational level through the rapid degradation of the polypeptide following its incorporation into the thylakoid. Degradation of the 24,500 Mr polypeptide in partially bleached (41 h) Euglena was demonstrated by two different experimental approaches involving isolated plastids and pulse-chase experiments using whole cells. Based on the results from the pulse-chase experiments it is estimated that the half-life of the 24,500 Mr species is 25 minutes in partially bleached cells. The same polypeptide, however, is maintained in control cultures at 33°C for at least 2 h. Furthermore, the activity responsible for the degradation of this polypeptide of the LHCPII appears to be selective since only the 24,500 Mr species is significantly degraded in isolated plastids from partially bleached cells during the 1 h incubation period at 33°C. Although the 24,500 Mr polypeptide is not degraded when unbleached cultures of Euglena are incubated at 33°C for at least 2 h, the 24,500 Mr species is degraded in partially bleached cells incubated at 23°C or at 33 °C (pulse-chase experiments). These observations suggest that the membrane environment in partially bleached thylakoids is different from the membrane environment in control thylakoids and does not contribute to the stable integration of the polypeptide into partially bleached thylakoids. The membrane environment in thylakoids from control cells, on the other hand, permits the stable integration and maintenance of the newly synthesized polypeptide for at least 2 h at 33°C. Events at the post-translational level have been implicated in the regulation of chloroplast polypeptides such as the large (Chen et aI., 1988) and small (Schmidt and Mishkind, 1983) subunit of ribulose 1,5-bisphosphate carboxylase, plastocyanin (Merchant and Bogorad, 1986), subunits of the chloroplast coupling factor (Biekmann and Feierabend, 1985), and the QB-binding protein (Mattoo et aI., 1984). The seemingly widespread occurrence of post-translational regulation suggests that events at this level of regulation play an important role in maintaining a precise stoichiometric balance of photosynthetic proteins especially those that are components of multimeric complexes and in the removal of damaged or dispensable proteins from plastids. Acknowledgements The author thanks Drs. G. W. Schmidt and F. G. Plumley for their generous gift of antibody to the LHCPII of Chlamydomonas reinhardii and Ms. Beverly Richey for typing this manuscript. This work was supported by a grant from the National Science Foundation (DCB-8715422).
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WILLIAM ORTIZ
BURCKHARDT, J., J. TELFORD, and M. L. BIRNSTIEL: Detection of labelled RNA species by contact hybridization. Nucleic Acid Res. 6,2963-2971 (1979). CHEN, Z., J. CHASTAIN, S. R. AL-ABED, R. CHOLLET, and R. J. SPREITZER: Reduced CO 2/0 2 specificity of ribulose-bisphosphate carboxylase/oxygenase in a temperature-sensitive chloroplast mutant of Chlamydomonas. Proc. Nat. Acad. Sci. USA 85, 4696-4699 (1988). CHUA, N. H.: Electrophoretic analysis of chloroplast proteins. Methods in Enzymo!. 69, 434-446 (1980). Ems, R. J.: Chloroplast proteins: synthesis, transport and assembly. Ann. Rev. Plant Physio!. 32, 111-137 (1981). HOLLOWAY, P. J. and M. J. DALLING: Modulation of the light-stimulated loss of polypeptides from isolated wheat thylakoids in vitro. J. Plant Physio!. 132,273-278 (1988). LASKEY, R. A. and D. MILLS: Quantitative film detection of 3H and J4C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341 (1975). MArroo, A. K., H. HOFFMAN-FALK, J. B. MARDER, and M. EDELMAN: Regulation of protein metabolism: coupling of photosynthetic electron transport to in vivo degradation of the rapidly metabolized 32-kilodalton protein of the chloroplast membranes. Proc. Nat. Acad. Sci. USA 81, 1380-1384 (1984). MERCHANT, S. and L. BOGORAD: Rapid degradation of apoplastocyanin in Cu(II)-deficient cells of Chlamydomonas reinhardtii. J. Bio!. Chern. 261, 15850-15853 (1986).
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