Regulation of accumulation of the major thylakoid polypeptides in Chlamydomonas reinhardtii y-1 at 25 degrees C and 38 degrees C

The amount of messenger RNA (mRNA) for polypeptides of the chlorophyll a/b-protein complex of thylakoid membranes in etiolated and greening cells of Chlamydomonas reinhardtii y-1 was examined by immunoprecipitation and electrophoresis of products of in vitro translation to determine at which stage production of these polypeptides is regulated. Cells grown 4 d in the dark at 25 degrees C contained small amounts of translatable mRNA for the major membrane polypeptides. Exposure of these etiolated cells to light, under conditions in which the membrane polypeptides accumulated, resulted in a significant increase in the quantity of the mRNA. In contrast, when etiolated cells were incubated for 1-2 h in the dark at 38 degrees C, translation assays indicated that mRNA for the membrane polypeptides became abundant. Moreover, the quantity of the mRNA did not increase when these cells subsequently were exposed to light. Therefore, at 38 degrees C the cellular level of the polypeptides is not regulated by synthesis of mRNA. The in vitro synthesized polypeptides, which were precipitated with antibodies prepared against the purified thylakoid polypeptides, had apparent molecular weights of 31,500 and 30,000. The corresponding immunoprecipitated polypeptides made in vivo had apparent molecular weights of 29,500 and 26,000. Thus, the membrane polypeptides are made as precursors. No net accumulation of the polypeptides occurred in cells in the dark at 38 degrees C, but immunoreactive polypeptides the size of the mature membrane components were labeled during incubation of cells with [14C]acetate in the dark. These results indicated that the mRNA was translated in the dark, but since the polypeptides did not accumulate, the products of translation were probably degraded. We conclude from our experiments that at 25 degrees C production of the polypeptides is regulated by the level of translatable mRNA in the cells. At 38 degrees C, however, the accumulation of the polypeptides is controlled by posttranslational processes.     

Accumulation of Chlorophyll a/b-Binding Polypeptides in Chlamydomonas reinhardtii y-1 in the Light or Dark at 38 degrees C : Evidence for Proteolytic Control

The kinetics of accumulation of light harvesting chlorophyll (Chl) a/b-binding polypeptides (LHCPs) in thylakoid membranes were analyzed during greening of Chlamydomonas reinhardtii y-1 at 38°C. Initial accumulation of LHCPs in thylakoid membranes was linear; LHCP precursors or polypeptides in transit within the chloroplast stroma were not detected. The rate of accumulation in the light was at least five-fold greater than that in the dark. The relatively small amount of LHCPs that accumulated in the dark was integrated properly in the membrane, as judged by the pattern of cleavage in vitro by exogenous proteases, and did not turn over at a significant rate in vivo. The kinetic data suggested that in y-1 cells either translation of LHCP mRNA was inhibited in the dark or newly synthesized polypeptides were degraded concurrently with transport into the chloroplast unless rescued by Chl. LHCPs accumulated in cells of the Chl b-deficient strain pg-113 at the same rate in the dark or the light at 38°C, an indication that light did not affect translation of LHCP mRNA. Membrane-associated LHCPs in pg-113 cells were completely degraded, in contrast to those in y-1 cells, by exogenous proteases, which suggested that pg-113 cells are deficient in a proteolytic activity. A peptidase was recovered from y-1 cells in a membrane fraction with a buoyant density slightly less than that of thylakoid membranes. Although a role for this activity in degradation of LHCPs has not been established, the specific activity of this peptidase in pg-113 cells was only 10 to 15% of the level in y-1 cells.     

A MAJOR POLYPEPTIDE OF CHLOROPLAST MEMBRANES OF CHLAMYDOMONAS REINHARDI : Evidence for Synthesis in the Cytoplasm as a Soluble Component

Electrophoresis of thylakoid membrane polypeptides from Chlamydomonas reinhardi revealed two major polypeptide fractions. But electrophoresis of the total protein of green cells showed that these membrane polypeptides were not major components of the cell. However, a polypeptide fraction whose characteristics are those of fraction c (a designation used for reference in this paper), one of the two major polypeptides of thylakoid membranes, was resolved in the electrophoretic pattern of total protein of green cells. This polypeptide could not be detected in dark-grown, etiolated cells. Synthesis of the polypeptide occurred during greening of etiolated cells exposed to light. When chloramphenicol (final concentration, 200 µg/ml) was added to the medium during greening to inhibit chloroplastic protein synthesis, synthesis of chlorophyll and formation of thylakoid membranes were also inhibited to an extent resulting in levels of chlorophyll and membranes 20–25% of those found in control cells. However, synthesis of fraction c was not affected by the drug. This polypeptide appeared in the soluble fraction of the cell under these conditions, indicating that this protein was synthesized in the cytoplasm as a soluble component. When normally greening cells were transferred from light to dark, synthesis of the major membrane polypeptides decreased. Also, it was found that synthesis of both subunits of ribulose 1, 5-diphosphate carboxylase was inhibited by chloramphenicol, and that synthesis of this enzyme stopped when cells were transferred from light to dark.     

Origin of Thylakoid Membranes in Chlamydomonas reinhardtii y-1 at 38 degrees C

The origin of thylakoid membranes was studied in Chlamydomonas reinhardtii y-1 cells during greening at 38°C. Previous studies showed that, when dark-grown cells are exposed to light under these conditions, the initial rates of accumulation of chlorophyll and the chlorophyll a/b-binding proteins in membranes are maximal (MA Maloney JK Hoober, DB Marks [1989] Plant Physiol 91: 1100-1106; JK Hoober MA Maloney, LR Asbury, DB Marks [1990] Plant Physiol 92: 419-426). As shown in this paper, photosystem II activity, which was nearly absent in dark-grown cells, also increased at a linear rate in parallel with chlorophyll. As compared with those made at 25°C, photosystem II units assembled during greening at 38°C were photochemically more efficient, as judged by saturation at a lower fluence of light and a negligible loss of excitation energy as fluorescence. Electron microscopy of cells in light for 5 or 15 minutes at 38°C showed that these initial, functional thylakoid membranes developed in association with the chloroplast envelope.     

Biogenesis of Thylakoid Membranes in Chlamydomonas reinhardtii y1 (A Kinetic Study of Initial Greening)

Initiation of thylakoid membrane assembly was examined in degreened cells of Chlamydomonas reinhardtii y1 cells depleted of thylakoid membranes and photosynthetic activity by growth in the dark for 3 to 4 d. Photoreductive activities of photosystem II (PSII) and photosystem I (PSI) increased with no apparent lag when degreened cells were exposed to light at 38[deg]C. However, fluorescence transients induced by actinic light, which reflect the functional state of PSII, changed only slightly during the first 2 h of greening. When these cells were treated with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) or saturating light, fluorescence increased commensurate with the cellular content of chlorophyll. In similar experiments with greening cells of C. reinhardtii CC-2341 (ac-u-g-2.3), a PSI-minus strain, fluorescence increased with chlorophyll without treatment with DCMU. These data suggested that fluorescence of initial PSII centers in greening y1 cells was quenched by activity of PSI. Continuous monitoring of fluorescence in the presence or absence of DCMU showed that assembly of quenched PSII centers occurred within seconds after exposure of y1 cells to light. These results are consistent with initial assembly of PSI and PSII within localized domains, where their proximity allows efficient energy coupling.     

Comparative analysis of the biogenesis of photosystem II in the wild- type and Y-1 mutant of Chlamydomonas reinhardtii

Expression of the genes of the photosystem II (PSII) core polypeptides D1 and D2, of three proteins of the oxygen evolving complex of PSII and of the light harvesting chlorophyll a/b binding proteins (LHCP) has been compared in wild-type (wt) and in the y-1 mutant of Chlamydomonas reinhardtii. Since wt, but not y-1 cells produce a fully developed photosynthetic system in the dark, comparison of the two has allowed us to distinguish the direct effect of light from the influence of plastid development on gene expression. The PSII core polypeptides and LHCP are nearly undetectable in dark-grown y-1 cells but they accumulate progressively during light induced greening. The levels of these proteins in wt are the same in the light and the dark. The amounts of the proteins of the oxygen evolving complex do not change appreciably in the light or in the dark for both wt and y-1. Steady state levels of chloroplast mRNA encoding the core PSII polypeptides remain nearly constant in the light or the dark and are not affected by the developmental stage of the plastid. Levels of nuclear encoded mRNAs for the oxygen evolving proteins and of LHCP increase during light growth in wt and y-1. In contrast to wt, synthesis of LHCP proteins is not detectable in y-1 cells in the dark but starts immediately after transfer to light, indicating that LHCP synthesis is controlled by a light-induced factor or process. While the rates of synthesis of D1 and D2 are immediately enhanced by light in wt, this increase occurs only after a lag in y-1 and thus must be dependent on an early light-induced event in the plastid. These results show that the biosynthesis of PSII is affected by light directly, by the stage of plastid development, and by the interaction of light and events associated with plastid development.     

Changes in thylakoid polypeptide phosphorylation during membrane biogenesis in Chlamydomonas reinhardii y-1

Thylakoid polypeptide phosphorylation has been studied in vivo and in vitro during plastid differentiation in Chlamydomonas reinhardii y-1. Pulse labeling cells at different stages of greening with [³²P]orthophosphate revealed differences in the pattern of protein phosphorylation. In the early phase of greening the 44–47 kDa reaction center II polypeptides were labeled but the 22–24 kDa polypeptides of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHC) were not. Later in the greening, coinciding with the formation of the antenna of Photosystem I and membrane stacking, the converse was found. Furthermore, the 22–24 kDa polypeptides of grana lamellae were less labeled than the same polypeptides found in the corresponding stroma lamellae. Polypeptides in the molecular mass range of 32–34 kDa were phosphorylated at all stages following the onset of greening. Dark-grown cells did not incorporate ³²P in vivo or in vitro into the polypeptides present in the residual thylakoids. Similarly, cells greened in the presence of chloramphenicol, in which the synthesis of reaction centers is inhibited, showed no light-stimulated phosphorylation in vitro. However, the residual 32–34 kDa and 44–47 kDa polypeptides found in thylakoids of these cells were phosphorylated in vivo, whereas the LHC polypeptides synthesized in the presence of chloramphenicol were not. Phosphorylation of the LHC polypeptides (22–24 kDa) in these cells occurred if new reaction center polypeptides and all antennae components were formed, following removal of the inhibitor and further incubation of the cells in the light. Phosphorylation of LHC polypeptides was not resumed if active reaction centers were formed in the absence of complete restoration of all antenna components (incubation in the dark or light with addition of cycloheximide). It is concluded that phosphorylation is correlated with the thylakoid polypeptide content and organization.     

Thylakoid membrane biogenesis in Chlamydomonas reinhardtii 137+. II. Cell-cycle variations in the synthesis and assembly of pigment

Synthesis of the chlorophyll and the major carotenoid pigments and their assembly into thylakoid membrane have been studied throughout the 12-h light/12-h dark vegetative cell cycle of synchronous Chlamydomonas reinhardtii 137+ (wild-type). Pulse exposure of cells to radioactive acetate under conditions in which labeling accurately reflects lipogenesis, followed by cellular fractionation to purify thylakoid membrane, allowed direct analysis of the pigment synthesis and assembly attendant to thylakoid biogenesis. All pigments are synthesized and assembled into thylakoids continuously, but differentially, with respect to cell-cycle time. Highest synthesis and assembly rates are confined to the photoperiod (mid-to-late G1) and support chlorophyll and carotenoid accretion before M-phase. The lower levels at which these processes take place during the dark period (S, M, and early-to- mid G1) have been ascribed to pigment turnover. Within this general periodic pattern, pigment synthesis and assembly occur in a "multi- step" manner, i.e., by a temporally-ordered, stepwise integration of the various pigments into the thylakoid membrane matrix. The cell-cycle kinetics of pigment assembly at the subcellular level mirror the kinetics of pigment synthesis at the cellular level, indicating that pigment synthesis not only provides chlorophyll and carotenoid for thylakoid biogenesis but may also serve as a critical rate-determinant to pigment assembly.     

Polypeptides of chloroplastic and cytoplastic origin required for development of Photosystem II activity,and chlorophyll-protein complexes,in Euglena gracilis Z chloroplast membranes

The development of photosynthetic activity and synthesis of chloroplast membrane polypeptides was studied during greening of Euglena gracilis Z in alternate light-dark-light cycles. The results show: (a) The development of both Photosystem II and Photosystem I can be dissociated from chlorophyll synthesis. (b) Most of the polypeptides required for development of Photosystem I are already synthesized during the initial light period (10–12 h); the further rise in Photosystem I activity in the dark is not inhibited by cycloheximide nor by chloramphenicol. (c) The development of Photosystem II requires continuous de novo synthesis of polypeptides and is inhibited by chloramphenicol. The water-splitting activity already present at the end of the first light period decays in the presence of chloramphenicol while that of 1,5-diphenylcarbazide oxidation is only partially retained. The activity can be repaired in the absence of chlorophyll synthesis and is correlated with the de novo synthesis of polypeptides of 50 000–60 000 daltons. The synthesis of these polypeptides and associated repair of Photosystem II activity is not inhibited by cycloheximide. (d) The chloroplast membranes can be resolved into about 40 distinct polypeptides, among them several in the molecular weight range 50 000–60 000, 20 000–35 000 and 10 000–15 000, which are major membrane constitutents. (e) The synthesis of two major polypeptides (M_r = 20 000–30 000) required for the formation of chlorophyll-protein complex(es) containing chlorophyll a and traces of chlorophyll b (CPII?) is light-dependent and cycloheximide-inhibited. It is concluded that the synthesis and addition to the growing membrane of chlorophyll and polypeptides required for the formation of Photosystem II and Photosystem I complexes can be dissociated in time. The H₂O-splitting enzyme(s) and possibly other components of Photosystem II complex are of chloroplastic origin and turn over in the dark while at least some of the chlorophyll binding polypeptides are of cytoplastic origin and their synthesis is light-controlled.     

Chloroplast-cytoplasmic interrelations involved in chloroplast development in Chlamydomonas reinhardi y-1: effect of selective depletion of chloroplast translates

Chlamydomonas reinhardi y-1 cells grown in the dark in the presence of chloramphenicol (CD cells) are depleted of photosynthetic membranes and 70S translates. These cells were found to be unable to synthesize chlorophyll in the light until chloroplast protein synthesis was resumed. On the other hand, CD cells acquired the capacity to partially green in the presence of cycloheximide. This greening was characterized by the development of photosynthetic activity, as demonstrated by light- dependent oxygen evolution of whole cells and by measurements of ribulose-1,5-bisphosphate carboxylase and fluorescence kinetics. The chlorophyll synthesized de novo during greening in the absence of 80S ribosomal activity was organized in chlorophyll-protein complexes, as ascertained by low-temperature fluorescence-emission spectra. The morphology of these cells appeared to be normal. A model has been proposed as a working hypothesis, which could account for the phenomena described above and previously reported data pertaining to chloroplast development.     

Chloroplast-encoded PsbT is required for efficient biogenesis of photosystem II complex in the green alga Chlamydomonas reinhardtii

The small hydrophobic polypeptide PsbT is associated with the photosystem II (PSII) reaction center (D1/D2 heterodimer). Here, we report the effect of the deletion of PsbT on the biogenesis of PSII complex during light-induced greening of y-1 mutants of the green alga Chlamydomonas reinhardtii. The y-1 is unable to synthesize chlorophylls in the dark but do so in the light. The dark-grown y-1 cells accumulated no major PSII proteins but a small amount of PsbT. Upon illumination, PsbT was immediately synthesized while chlorophylls, major PSII proteins, and O(2)-evolving activity increased after a 1-h lag. The y-1 cells without PsbT accumulated chlorophylls and PSI protein at a similar rate, whereas the accumulation of PSII complex was specifically retarded during greening. The absence of PsbT did not affect the synthesis of PSII proteins. These results indicate that PsbT is required for the efficient biogenesis of PSII complex.     

BIOGENESIS OF CHLOROPLAST MEMBRANES : IV. Lipid and Pigment Changes during Synthesis of Chloroplast Membranes in a Mutant of Chlamydomonas reinhardi y-1

The glycolipid, phospholipid, pigment, and fatty acid content in whole y-1 cells during the greening process have been investigated. The time course of their changes indicates that phosphatidyl glycerol and glycolipids are the main lipids synthesized specifically during illumination of dark-grown cells, concomitant with an increase in the polyunsaturated C18:2 and C18:3 fatty acids. The pigment complex of light-grown cells consists mainly of chlorophylls a and b, lutein, β-carotene, violaxanthin, and neoxanthin. During the greening process, chlorophylls a and b are synthesized in constant proportions (ratio a/b equals 2.6), β-carotene and violaxanthin do not change significantly, and lutein and neoxanthin increase. The molar ratios of the different lipids and pigment to total chlorophyll during greening has been calculated. It was found that during the initial phase of greening when chlorophyll is synthesized at increasing rates, the molar ratios of various lipids and pigments to chlorophyll decrease and tend to become constant when chlorophyll and membrane synthesis proceed at constant rates. The implication of these findings with respect to the concept of membrane assembly through a spontaneous single step process is discussed     

BIOGENESIS OF CHLOROPLAST MEMBRANES : II. Plastid Differentiation during Greening of a Dark-Grown Algal Mutant (Chlamydomonas reinhardi)

Dark-grown cells of the y-1 mutant of Chlamydomonas reinhardi contain a partially differentiated plastid lacking the photosynthetic lamellar system. When exposed to the light, a rapid synthesis of photosynthetic membranes occurs accompanied by synthesis of chlorophyll, lipids, and protein and extensive degradation of the starch reserve. The process is continuously dependent on illumination and is completed within 6–8 hr in the absence of cell division. Photosynthetic activity (O₂ evolution, Hill reaction, NADP photo-reduction, and cytochrome f photooxidation) parallels the synthesis of pigment and membrane formation. During the greening process, only slight changes occur in the levels of soluble enzymes associated with the photosynthetic process (RuDP-carboxylase, NADP-linked G-3-P dehydrogenase, alkaline FDPase (pH 8)) as compared with the dark control. Also cytochrome f concentration remains almost constant during the greening process. The kinetics of the synthesis of chlorophyll, formation of photosynthetic membranes, and the restoration of photosynthetic activity suggest that the membranes are assembled from their constituents in a single-step process.     

Localization of light-harvesting complex apoproteins in the chloroplast and cytoplasm during greening ofChlamydomonas reinhardtii at 38°C

Assembly of the major light-harvesting complex (LHC II) and development of photosynthetic function were examined during the initial phase of thylakoid biogenesis inChlamydomonas reinhardtii cells at 38°C. Continuous monitoring of LHC II fluorescence showed that these processes were initiated immediately upon exposure of cells to light. However, mature-size apoproteins of LHC II (Lhcb) increased in amount in an alkali-soluble (non-membrane) fraction in parallel with the increase in the membrane fraction. Alkali-soluble Lhcb were not integrated into membranes when protein synthesis was inhibited, suggesting that they were not active intermediates in LHC II assembly, nor were they recovered in a purified chloroplast preparation. Immunocytochemical analysis of greening cells revealed Lhcb inside the chloroplast near the envelope and in clusters deeper in the organelle. Antibody binding also detected Lhcb in granules within vacuoles in the cytosol, and Lhcb were recovered in granules purified from greening cells. Our results suggest that the cytosolic granules serve as receptacles of Lhcb synthesized in excess of the amount that can be accommodated by thylakoid membrane formation within the plastid envelope.     

Heat shock response ofChlorella protothecoides during greening

Heterotrophically grown cells ofChlorella protothecoides were transferred to autotrophic medium and allowed to green at 25°C. The protein synthetic activity of the greening cells measured in terms of incorporation of [³5S]-methionine showed a maximum around 20 h of greening and thereafter started declining. Similarly, an analysis of densitometric tracings of the fluorographic profile of the polypeptides associated with both total cellular fraction and membrane fractions during different hours of greening revealed that maximum number of polypeptides were getting labelled around 20 h of greening. At 20 h of greening, the cells were shifted to 40°C and the effect of heat shock on protein synthesis was studied. The heat shock treatment caused a definite decrease in the incorporation of [³⁵S]-methionine into proteins. Due to heat shock, the synthesis of total soluble proteins was affected much more than that of the thylakoid membrane bound proteins. When the cells were transferred back to 25°C after a brief period of heat shock at 40°C, there was a considerable recovery in the protein synthesis and this recovery was found to be significant in the case of soluble proteins, while there was no such definite recovery in the synthesis of thylakoid membrane bound proteins.     

Studies on chloroplast development in Chlamydomonas reinhardtii I. Effect of brief illumination on chlorophyll synthesis

When dark grown cells of Chlamydomonas reinhardtii y-1 mutantwere exposed to continuous light, an immediate transformationof small amounts of protochlorophyll(ide), which had been presentin the dark grown cells, to chlorophyll was observed. Afterthis, there was a slow accumulation of chlorophyll lasting for2.5-3 hr before the start of exponential synthesis. Initialaccumulation of chlorophyll was distinctly slower at a highlight intensity (13,000 lux) than it was at moderate intensitiesof light (2,000–5,000 lux). However, the exponential synthesisof chlorophyll started after the same 2.5–3 hr of illumination. A brief pre-illumination of cells followed by incubation indarkness was effective in promoting chlorophyll synthesis undersubsequent continuous illumination at high, as well as moderatelight intensities. Pretreatment alleviated retardation of theinitial chlorophyll accumulation by light of high intensity.The promoting effect of preillumination on chlorophyll synthesiswas sufficient, even when a light impulse as short as 10 secwas given. However, the effect was dependent on length of thedark period after the short pre-illumination. The full extentof this effect was observed when the dark period was about 2.5–3hr long. Further dark incubation gradually decreased the effect. On the basis of these findings, it is assumed that a factor(s)responsible for promotion of chlorophyll (or chloroplast) synthesisin the process of greening of dark grown cells is produced duringthe dark period after a brief pre-illumination, and that thefactor is turned over at a relatively fast rate. The possiblenature of the presumed factor is discussed in relation to chloroplastdevelopment. ¹Present address: Department of Biology, Faculty of Science,Kobe University, Nada-ku, Kobe, Japan. (Received August 18, 1970; )     

Synthesis of chloroplast membrane polypeptides during synchronous growth of Chlamydomonas reinhardtii

The synthesis of the major chloroplast membrane polypeptides has been studied during synchronous growth of Chlamydomonas reinhardtii. Under these conditions, chlorophyll is synthesized during the latter part of the light period and cell division takes place during the dark period. The profile of the chloroplast membrane polypeptides of C. reinhardtii has been well characterized and shown to contain two major classes by size (Hoober, J. 1970. J. Biol. Chem. 245:4327). Polypeptides of group I have a mol wt range of 50,000–55,000 daltons. The second region consists of at least three polypeptide groups, IIa, IIb, and IIc, having mol wt of 40,000, 31,000, and 27,000 daltons, respectively. The synthesis of these polypeptides has been measured using a double-labeling technique and a computer-aided statistical analysis. The rate of labeling of group I polypeptides is highest during the early light period and decreases after 6 h of growth. Group IIa is labeled from the beginning of the light period, but little synthesis of IIb occurs before 3 h, and significant amounts of label are not found in IIc before 5 h of growth. After approximately 8 h of light, groups IIb and IIc are synthesized at rates significantly greater than those of the other membrane polypeptides. The synthesis of the major polypeptide groups ceases in the dark. We conclude that the biosynthesis of the chloroplast membranes is a sequential or stepwise process.     

Kinetics of Accumulation of Ribulose-1,5-bisphosphate Carboxylase during Greening in Euglena gracilis: Photoregulation

The preparation of a rabbit antibody to ribulose-1,5-bisphosphate carboxylase (RuBPCase) from Euglena gracilis and its use to quantitate RuBPCase in dark- and light-grown cells and during light-induced chloroplast development (greening) are described. Light-grown Euglena have at least 36 times more RuBPCase than dark-grown Euglena. Light is required for both the initiation and continued increase in net synthesis of RuBPCase over the dark level: brief illumination 12 hours before exposure to continuous light eliminates the lags in the accumulation and increase in activity of RuBPCase (as well as in chlorophyll accumulation); net synthesis is blocked in greening cells returned to the dark or exposed to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Streptomycin or cycloheximide prevents RuBPCase accumulation when added at the beginning of greening but only partially blocks accumulation when added after 25 hours of greening. After 24 hours of greening, the activity of RuBPCase per milligram chlorophyll continues to increase slowly while concentration of the enzyme per milligram chlorophyll remains constant. This increased activity may be due to activation of the enzyme as well as to net synthesis.     

LIGHT-HARVESTING COMPLEX AF'OPROTEINS IN CYTOPLASMIC VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Cells of Chlamydomonas reinhardtii Dangeard strain cw15arg7A contain electron-opaque material, often in the form of large granules, within cytoplasmic vacuoles. Immunoelectron microscopy with antibodies to polypeptide 11, a component of the major light-harvesting chlorophyll (Chl) a/b-protein complex (LHCII,) of thylakoid membranes, revealed the presence of LHCII Polypeptides within the chloroplast and in vacuolar material in cells grown in the light. Vacuolar material was also heavily immunodecorated in dark-grown cells that did not synthesize Chl. Accumulation of LHCII polypeptides was further studied in greening and light-grown cells of a pale green mutant, deficient in LHCII, that was derived from cu15arg7A by insertional mutagenesis. Light-grown cells of this mutant strain contained relatively few thylakoid membranes and synthesized LHCII polypeptides at a low rate. However, cytoplasmic vacuoles were immunoreactive. Appearance of mature-sized LHCII polypeptides in vacuoles suggested that these proteins were partially translocated across the envelope but not retained by the chloroplast without assembly of LHCII.