The lithium-chloride-soluble cell-wall layers of Chlamydomonas reinhardii contain several immunologically related glycoproteins

Cell-wall glycoproteins of the unicellular green alga Chlamydomonas reinhardii have been purified from LiCl extracts of intact cells by gel exclusion chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies were raised against several polypeptide components isolated from the LiCl extracts. All these antibodies specifically reacted with the cell surface of formaldehyde-fixed cells. They showed cross-reactivity with the different antigens and were also reactive against some other polypeptides present in the LiCl extracts of intact wild-type cells as shown by double-diffusion assays and immunoblot analyses. These antigens were largely missing in LiCl extracts from the cell-wall-deficient mutant CW-15. The pattern of immunologically related cell-wall polypeptides of C. reinhardii varied during the vegetative cell cycle and was found to be also dependent on the growth conditions. Dot-immunobinding assays on chemically modified cell-wall glycoproteins demonstrated differences between the various antibodies with respect to their specificities. Differences were observed especially with respect to their reactivities against chemically deglycosylated cell-wall polypeptides. Chemical deglycosylation generally reduced the binding of the different antibodies indicating that all these antibodies recognize carbohydrate side chains. Only two of these antibody preparations, raised against cell-wall glycoproteins of relative molecular mass 35 and 150 kilodaltons, were found to be strongly reactive against deglycosylated cell-wall polypeptides. When these antibodies were saturated with cell-wall-derived glycopeptides in order to abolish the binding to carbohydrate side chains, they still recognized the same cell-wall polypeptides as did the untreated antibodies. These findings indicate that the cross-reactivity of the different cell-wall polypeptides with the antibodies is not exclusively the consequence of similar glycosylation patterns but is also the result of the presence of similar structures within the non-glycosylated stretches of the polypeptide backbones. Cell walls isolated from growing tobacco pollen tubes contained a single polypeptide component which showed crossreactivity with the antibodies to the cell-wall glycoproteins of C. reinhardii.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - kDa kilodalton - M_r relative molecular mass - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Cell wall glycoproteins from Chlamydomonas reinhardii,and their self-assembly

We have investigated the in vitro reassembly of the salt soluble, hydroxyproline rich, glycoproteins from the cell wall of Chlamydomonas reinhardii, into structured cell wall fragments. We have devised an assay which has been used to follow the reassembly of the unfractionated and fractionated (2BI and 2BII) cell wall glycoproteins. Reassembly has a pH optimum of 5, a temperature optimum of 20°C, and the final size of the reassembled fragments appears to be promoted by the minor component 2BI. Periodate oxidation experiments show that sugar residues, in particular mannose, are important for accurate reassembly. Using electron microscopy, the structure of the reassembled products has been elucidated, as have intermediate stages in the reassembly process.Abbreviations TRIS Tris(hydroxymethyl)-methylamine - SDS Sodium dodecyl sulphate - PAGE Polyacrylamide gel electrophoresis This is the fifth paper in a series entitled Structure composition and morphogenesis of the cell wall of Chlamydomonas reinhardii. The last paper in this series was Catt et al. (1976)     

Assembly of cell-wall glycoproteins of Chlamydomonas reinhardii: Oligosaccharides are added in medial and trans Golgi compartments

A series of monoclonal antibodies and a polyclonal antiserum have been used to investigate the localisation and pathway of biosynthesis of the cell-wall hydroxyproline-rich glycoprotein 2BII in the alga Chlamydomonas reinhardii. Glyco-protein precursors were detected within the endoplasmic reticulum using a polyclonal antiserum raised to the deglycosylated 2BII. Monoclonal antibodies which are known to recognise different carbohydrate epitopes of 2BII were found to label two distinct regions of the Golgi stack. The immunolabelling results demonstrate that there is compartmentation of protein synthesis and glycosylation steps for these O-glycosidically linked glycoproteins. Newly synthesised glycoproteins are transported from the Golgi apparatus to the cell surface via two distinct routes. They then undergo assembly into a cell wall, the inner wall layer being formed first and probably functionaing as a template within which the outer crystalline wall layers are assembled.Abbreviations DGP deglycosylated glycoprotein - ER endoplasmic reticulum - MAC monoclonal antibody centre - M _r relative molecular mass     

Identification of a highly conserved hydroxyproline-rich glycoprotein in the cell walls of Chlamydomonas reinhardtii and two other Volvocales

The unicellular alga Chlamydomonas reinhardtii Dang, has a cell wall made entirely from hydroxyproline-rich glycoproteins (HRGPs). We recently employed a quantiative in vitro reconstitution system (Adair et al. 1987, J. Cell Biol. 105, 2373–2382) to assign outer-wall HRGPs of C. reinhardtii to specific sublayers, and describe the major interactions responsible for their assembly. Some of these interactions appear to involve relatively conserved HRGP domains, as evidenced by interspecific cell-wall reconstitution between C. reinhardtii and two multicellular Volvocales (Volvoxcarteri lyengar and Gonium pectorale Müller). In the present report we provide biochemical and immunological evidence that the outer cell-walls of V. carteri and G. pectorale both contain prominent HRGPs closely related to C. reinhardtii GP2. Identification of conserved GP2 homologues indicates a molecular basis for interspecific reconstitution and provides a useful avenue for characterization of HRGP domains mediating cell-wall formation in these algae.Abbreviations GP1, 2, 3 outer-cell wall glycoproteins 1, 2, and 3 - GP2_dg deglycosylated GP2 - HRGP hydroxyprolinerich glycoprotein - SDS-PAGE sodium docecyl sulfate polyacrylamide gel electrophoresis     

Variability of mitochondrial population in Chlamydomonas reinhardii

Several details have been published cocerning the mitochondrial number and shapes at various stages of the synchronized vegetative and generative cell cycle in Chlamydomonas reinhardii. The present study, based on ultrathin serial sections and threedimensional reconstructions, completes these data. Quantitative analysis of serial micrographs makes it possible to give specific details of mitochondrial volumes in cells at early intermediate stages of the vegetative life cycle. Our investigations clearly show that mitochondria have a relatively wide range of sizes, within certain limits, and vary like the mitochondrial shapes; in fact, they vary in various cells at various stages as well as in several cells at the same stage and even in one and the same cell. Thus, we present a plastic insight into the dynamically changing micromorphology of the mitochondrial population in Chlamydomonas reinhardii.     

Purification and molecular properties of ferredoxin-glutamate synthase from Chlamydomonas reinhardii

Ferredoxin-glutamate synthase (EC 1.4.7.1) from Chlamydomonas reinhardii has been purified to electrophoretic homogeneity, with a specific activity of 10.4 units mg^-1 protein, by a method which included chromatography on diethylaminoethyl sephacel and hydroxylapatite, and ferredoxin-sepharose affinity treatment. The enzyme is a single polypeptide chain of M _r 146000 dalton which shows an absorption spectrum with maxima at 278, 377 and 437 nm, and an A₂₇₆/A₄₃₇ absorptivity ratio of 7.0. The anaerobic addition of dithionite results in the loss of the absorption peak at 437 nm, which is restored upon reoxidation of the enzyme with an excess of 2-oxoglutarate, alone or in the presence of glutamine. This indicates the presence in the enzyme of a flavin prosthetic group, which is functional during the catalysis. The ferredoxin-glutamate synthase can be assayed with methyl viologen, chemically reduced with dithionite, but it is unable to use reduced pyridine nucleotide. Azaserine, 6-diazo-5-oxo-norleucine, bromocresol green and p-hydroxymercuribenzoate are potent inhibitors of this activity, which, on the other hand, is stable upon heating at 45°C for 10 min.Abbreviations DEAE-sephacel diethylaminoethyl sephacel - Fd ferredoxin - GOGAT glutaniate synthase (glutamine: -ketoglutarate aminotransferase) - SDS sodium dodecyl sulfate     

Biosynthesis of ferredoxin in Chlamydomonas reinhardii

The selective action of the antibiotics chloramphenicol and cycloheximide on the synthesis of ferredoxin in liquid cultures of Chlamydomonas reinhardii was studied. Highly specific antibodies raised against Chlamydomonas ferredoxin were used to determine the in vivo synthesis of apoferredoxin and conversion into native protein. The results indicate that 80S ribosomes are involved in the synthesis. Chlamydomonas cells growing in the absence of iron did not synthesize immunologically detectable amounts of ferredoxin. We suggest that this is based upon feed-back inhibition of apoferredoxin synthesis at the translational level.Abbreviations CAP chloramphenicol - CHI cycloheximide - IgG Immunoglobulin G - PBS 140.4 mM NaCl. 9 mM Na₂HPO₄, 1.3 mM NaH₂PO₄ (pH 74) - SDS sodium dodecvl sulphate - Fd Ferredoxin - apoFd Apoferredoxin - CM-Fd Scarboxymethylated Fd - TCA-Fd Fd treated with trichloroacetic acid     

Isolation and characterization of prolyl hydroxylase from Chlamydomonas reinhardii

Prolyl hydroxylase, which is responsible for the hydroxylation of peptidyl proline residues, has been isolated and purified from the green alga Chlamydomonas reinhardii. The enzyme, which appears to be loosely associated with microsomal membranes, was released into solution by sonication in the presence of detergent. Purification was achieved by ion-exchange chromatography followed by affinity chromatography using the immobilized substrate poly-L-proline. Apart from its differing substrate specificity the enzyme appears to possess similar molecular characteristics to prolyl hydroxylase isolated from animal tissues: the active enzyme is a tetramer of about 240–250 kDa and nonidentical monomers of 65 and 60 kDa. The monomers are capsule shaped having a dimension of 12×7 nm.Abbreviations Da dalton - DEAE diethylaminoethyl - DTT dithiothreitol - Hepes 4-(2-hydroxymethyl)-1-piperazine ethanesulfonic acid - -KGA -ketoglutarate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Isolation and characterization of an extracellular hydroxyproline-rich glycoprotein and a mannose-rich polysaccharide from Eudorina californica (Shaw)

Mucilage and colony walls of E. californica were separated from the cells by homogenization, filtration, and differential centrifugation. The chief components of the mucilage were a high-molecular-weight (MW) hydroxyproline-rich glycoprotein and a very high-MW polysaccharide in the proportions 47% and 34%, respectively. The glycoprotein consisted of galactose, arabinose, xylose and an unidentified neutral sugar; and the amino acids cysteine, aspartic acid, glutamic acid, arginine, lysine, glycine, serine, methionine, histidine, alanine, proline, hydroxyproline, tyrosine, threonine, valine, phenylalanine, isoleucine and leucine. The principal sugar of the polysaccharide was mannose. The chemical composition of the colony walls was essentially the same as that of the glycoprotein in the mucilage except that there was almost twice as much hydroxyproline. Also the protein content of the colony walls was 34% while that of the glycoprotein in the mucilage was 22%. No glucose, sugar acids or nucleic acids were found in the extracellular matrix.     

Ammonium generation by nitrogen-starved cultures of Chlamydomonas reinhardii

Ammonium (NH ₄ ⁺ ) assimilation by Chlamydomonas reinhardii was inhibited when cultures were incubated with methionine sulphoximine (MSO). Methionine sulphoximine inhibited glutamine synthetase acitvity in vitro in extracts from wild-type (2192) and mutant (CC419) cultures. Mutant cultures were insensitive to MSO inhibition in vivo. Nitrogen-starved, wild-type cultures excreted ammonium when they were incubated with MSO in light or in darkness. Ammonium generation was stimulated by glutamine, inhibited by CO₂ and stoichiometrically related to loss of protein. Notrogen replete cultures treated with MSO excreted ammonium in light but little was excreted in darkness. Ammonium excretion in darkness, in the presence of MSO, was enhanced by either a period of nitrogen deprivation or by the addition of acetate. Nitrogen deprivation also diminished the lag before ammonium excretion commenced.Abbreviation MSO methionine sulphoximine     

Transfer of organelles of the alga Chlamydomonas reinhardii into carrot cells by protoplast fusion

A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.Abbreviations PEG polyethylene glycol - TEM transmission electron microscopy - SEM scanning electron microscopy This study was undertaken during sabbatical leave in The Research School of Biological Sciences. Australian National University     

A model of carbon dioxide assimilation in Chlamydomonas reinhardii

A simple model of photosynthetic CO₂ assimilation in Chlamydomonas has been developed in order to evaluate whether a CO₂-concentrating system could explain the photosynthetic characteristics of this alga (high apparent affinity for CO₂, low photorespiration, little O₂ inhibition of photosynthesis, and low CO₂ compensation concentration). Similarly, the model was developed to evaluate whether the proposed defects in the CO₂-concentrating system of two Chlamydomonas mutants were consistent with their observed photosynthetic characteristics. The model treats a Chlamydomonas cell as a single compartment with two carbon inputs: passive diffusion of CO₂, and active transport of HCO ₃ ^- . Internal inorganic carbon was considered to have two potential fates: assimilation to fixed carbon via ribulose 1,5-bisphosphate carboxylase-oxygenase or exiting the cell by either passive CO₂ diffusion or reversal of HCO ₃ ^- transport. Published values for kinetic parameters were used where possible. The model accurately reproduced the CO₂-response curves of photosynthesis for wild-type Chlamydomonas, the two mutants defective in the CO₂-concentrating system, and a double mutant constructed by crossing these two mutants. The model also predicts steady-state internal inorganic-carbon concentrations in reasonable agreement with measured values in all four cases. Carbon dioxide compensation concentrations for wild-type Chlamydomonas were accurately predicted by the model and those predicted for the mutants were in qualitative agreement with measured values. The model also allowed calculation of approximate energy costs of the CO₂-concentrating system. These calculations indicate that the system may be no more energy-costly than C₄ photosynthesis.Abbreviations Chl chlorophyll - RuBPC/O ribulose 1,5-bisphosphate carboxylase-oxygenase - CA carbonic anhydrase     

Cell wall regeneration by protoplasts of Chlamydomonas

Protoplasts from Chlamydomonas smithii prepared by the action of C. reinhardii gamete autolysine have been studied with respect to cell wall regeneration. Natural protoplasts within sporangia were also investigated for purposes of comparison. In both cases a new cell wall is completed within 2–3 h of the onset of regeneration. The first visible stages of wall regeneration are to be seen after 40–60 min as a fine fringe outside of the plasmalemma. The development of the typical central triplet follows within the next 1 h. Cell wall regeneration is reversibly inhibited by cycloheximide (10g ml^-1) and reversibly disturbed by concanavalin A (50 g ml^-1). Actinomycin D at concentration over 100g ml^-1 also inhibit but the inhibition is irreversible and peculiar membrane effects are observed. Chelators (ethylenediamine tetraacetic acid; ethyleneglycol-bis-aminoethyl ether) and 2-deoxyglucose slightly retard or have no effect on cell wall regeneration.Abbreviations EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis(aminoethyl ether) - N,N tetraacetic acid     

Cell-wall abnormalities of a Chlamydomonas reinhardtii mutant strain under suboptimal growth conditions

Sporangia were accumulated in autotrophically and mixotrophically growing cultures of the Chlamydomonas reinhardtii mutant strain ls entering the stationary phase. Such an accumulation of sporangia was never observed in stationary-phase cultures of wildtype strains. Sporangia harvested from stationary-phase cultures of the mutant strain ls released their zoospores after being resuspended in fresh culture medium. Liberation of zoospores was also observed during fixation of these sporangia with glutaraldehyde and OsO₄. Release of zoospores during fixation was prevented by pretreatment with 3 mol·l^–1 LiCl. Ultrastructural analyses of these LiCl-pretreated sporangia revealed that they contained abnormal sporangial walls: sporangia containing sporangia and sporangia surrounded by additional multilayered cell walls have been observed. Similar abnormal cell-wall structures were found in sporangia accumulated at the end of the dark period, when the mutant strain ls was grown photoautotrophically under a 12 h light-12 h dark regime with suboptimal aeration. When grown under optimal conditions, this particular mutant did not show any abnormal wall structures.This work has been supported by a grant from the Deutsche Forschungsgemeinschaft. The authors thank Mrs. C. Adami for the photographic work.     

Sexual agglutination factor from Chlamydomonas eugametos

Chlamydomonas eugametos gametes can sexually agglutinate via their flagellar surfaces whereas vegetative cells cannot. Therefore, flagellar glycoproteins, present in gamete cells but absent from vegetative cells, were investigated as prospective mt ^-agglutination factors. They were identified as periodic acid Schiff (PAS) stained bands separated in sodium dodecyl sulphate-polyacrylamide electrophoresis gels. Gamete-specific bands were determined by comparison with equivalent gels of vegetative flagella and by immunological techniques using antisera raised against isolated mt ^- gamete flagella. Four high molecular weight flagellar glycoproteins proved to be gamete specific (PAS-1.2, PAS-1.3, PAS-3 and PAS-4). They were extracted from flagella by 3 M guanidine thiocyanate, separated in a column of Sepharose 2B, and tested for in vitro agglutination activity on mt ⁺ gametes. A single peak of activity was found to be correlated with the presence of the PAS-1.2 band. It is shown that mt ^- agglutination activity is related to the concentration of this glycoprotein in flagellar membranes.Abbreviations SDS sodium dodecyl sulphate - PAS periodic acid Schiff - GTC guanidine thiocyanate - mt ^-/+ mating type plus or minus     

Monoclonal antibodies to the major structural glycoprotein of the Chlamydomonas cell wall

A family of monoclonal antibodies (McAb) has been raised to the major cell-wall structural glycoprotein of the green alga Chlamydomonas reinhardii. The work represents an approach using McAb to the structure and function of plant cell wall-components. On the basis of cross-competition assays, the McAb have been subdivided into six groups. Various lines of evidence indicate that some of these group's e.g. group III, contain McAb which recognise specific oligosaccharide side chains of the glycoprotein. Heterogeneity of the oligosaccharide side chains is demonstrated, by probing Western blots of separated glycoproteins and glycopeptides with the McAb. The results are discussed in relation to the possibility of raising McAb as probes for the structure and function of higher-plant cell-wall oligosaccharides, which are increasingly being shown to be important in cell-cell recognition phenomena and host-pathogen interactions.Abbreviations McAb monoclonal antibody (bodies) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Occurrence of xanthine dehydrogenase in Chlamydomonas reinhardii: A common cofactor shared by xanthine dehydrogenase and nitrate reductase

Wild-type Chlamydomonas reinhardii cells have xanthine dehydrogenase activity when grown with nitrate, nitrite, urea, or amino acid media. Mutant strains 102, 104, and 307 of Chlamydomonas, lacking both xanthine dehydrogenase and nitrate reductase activities, were incapable of restoring the NADPH-nitrate reductase activity of the mutant nit-1 of Neurospora crassa, whereas wild type cells and mutants 203 and 305 had xanthine dehydrogenase and were able to reconstitute the nitrate reductase activity of nit-1 of Neurospora. Therefore, it is concluded that in Chlamydomonas a common cofactor is shared by xanthine dehydrogenase and nitrate reductase. Xanthine dehydrogenase is repressed by ammonia and seems to be inessential for growth of Chlamydomonas.     

Glycoprotein conformation in plant cell walls

The major structural glycoprotein of the cell wall of Chlamydomonas reinhardii has a protein core, at least 50% of which is in the unusual polyproline II conformation. This has been demonstrated by examining the circular dichroism of the cell wall, its constituent glycoproteins, and thermolysin released wall glycopeptides. One of these glycopeptides, T2, has a high hydroxyproline and sugar content, and possesses upward of 85% polyproline II structure. The main extracellular matrix glycoprotein therefore has a rigid, rod-like structure and the significance of this and its relation to higher plant cell wall glycoproteins is discussed. The unusual conformation appears to confer great stability on the glycoprotein as it is unchanged either by certain denaturing agents or during the transition from protomer to assembled cell wall.Abbreviations CD circular dichroism - HP 4-hydroxy-L-proline - PP poly-L-proline - SDS sodium dodecylsulphate This is the eight paper in a series entitled Structure, Composition and Morphogenesis of the Cell Wall of Chlamydomonas reinhardii. The last paper in this series was Catt et al. (1978)     

The cell wall of Chlamydomonas reinhardtii gametes: Composition,structure and autolysin-mediated shedding and dissolution

The cell walls of Chlamydomonas gametes are multilayered structures supported on frameworks of polypeptides extending from the plasma membrane. The wall-polypeptide catalogue reported by Monk et al. (1983, Planta 158, 517–533) and extended by U.W. Goodenough et al. (1986, J. Cell Biol. 103, 405–417) was re-evaluated by comparative analysis of mechanically isolated cell walls purified from several strains. The extracellular locus of wall polypeptides was verified by in vivo iodogen-catalysed iodination and by autolysin-mediated elimination of the bulk of these polypeptides from the cell surface. Three (w15, w16, w17) and possibly four (w14) polypeptides were located to the most exterior aspect of the wall because of their susceptibility to Enzymobeadcatalysed iodination and their retention by a cell-wall-less mutant. The composition of shed walls stabilised with ethylenediaminetetraacetic acid during natural mating and kinetic analysis of the dissolution of walls purified from a bald-2 mutant demonstrated the rapid and specific destruction of polypeptide w3. Differential solubilisation of wall polypeptides occurred after loss of w3. Wall dissolution, characterised by the generation of fishbone structures from the W2 layer, gave as many as four additional polypeptides. Charged detergents and sodium perchlorate extracted a comparable range of polypeptides at room temperature from mechanically isolated walls, i.e. components of the W4–W6 layers, hot sodium dodecyl sulphate solubilised framework polypeptides, while reducing agent was required to solubilise the W2 layer. A model of wall structure is presented.Abbreviations DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol