In vitro identification of rhodopsin in the green alga Chlamydomonas.

The unicellular alga Chlamydomonas can detect both intensity and direction of the ambient light and adjust its swimming speed and direction accordingly. On the basis of physiological experiments, the functional photoreceptor for this visual process has recently shown to be a rhodopsin. We here report the in vitro identification of endogenous retinal and a rhodopsin in Chlamydomonas cell extracts and purified membrane preparations. The rhodopsin absorption spectrum has fine structure with the maximum at 495 nm and matches the action spectra for the behavioral light responses. The rhodopsin can be bleached and subsequently reconstituted with exogenous retinal. Labeling with [3H]retinal occurs in the final preparation only with a single protein with a molecular weight of 32,000. We conclude that this protein is the visual photoreceptor in Chlamydomonas.

The proximal portion of Chlamydomonas flagella contains a distinct set of inner dynein arms.

A specific type of inner dynein arm is located primarily or exclusively in the proximal portion of Chlamydomonas flagella. This dynein is absent from flagella less than 6 microns long, is assembled during the second half of flagellar regeneration time and is resistant to extraction under conditions causing complete solubilization of two inner arm heavy chains and partial solubilization of three other heavy chains. This and other evidence described in this report suggest that the inner arm row is composed of five distinct types of dynein arms. Therefore, the units of three inner arms that repeat every 96 nm along the axoneme are composed of different dyneins in the proximal and distal portions of flagella.

[Presence of arrestin (S-antigen)-like proteins in vegetable cells]. / Présence de protéines apparentées à l'arrestine (antigène-S) dans des cellules végétales.

Arrestin (or S-antigen) is a protein that regulates phototransduction in photoreceptor cells of the retina. Homologous proteins have been recently detected in other, non-photosensitive, cells of vertebrates, where they are thought to be associated with other systems of signal transduction. Proteins crossreactive with retinal arrestin were detected in soluble cell extracts from Nicotiana tabacum and Chlamydomonas reinhardtii by immunoblotting using several antibodies against arrestin. Variations of the immunoreactive protein pattern were associated with the growth cycle of tobacco cells. These observations suggest that analogs of arrestin exist in the vegetal kingdom, where they could be involved in transduction processes.

Localization of an intermediate chain of outer arm dynein by immunoelectron microscopy.

We have used immunoelectron microscopy to determine the location of an intermediate chain in the isolated outer arm dynein from Chlamydomonas flagella. When the purified alpha beta dimer of the outer arm was incubated with antibodies recognizing two distinct epitopes on its 69-kDa intermediate chain and then negatively stained and examined by electron microscopy, both antibodies appeared to have bound to the base of the Y-shaped stem that connects the two heads of the particle. These results indicate that this intermediate chain is located at the base of the stem. Inasmuch as this polypeptide is tightly associated with the 78-kDa intermediate chain and several light chains in an intermediate chain-light chain complex, it is likely that this entire assemblage is located at the base of the particle. Thus, these polypeptides are in a potentially important position with regard to the ATP-insensitive (structural end) binding of dynein to microtubules and to dynein-dynein interactions within the axoneme.

Ubiquitin in Chlamydomonas reinhardii. Distribution in the cell and effect of heat shock and photoinhibition on its conjugate pattern.

Ubiquitin, a highly conserved 76-amino-acid protein, is involved in the response of many types of eukaryotic cells to stress but little is known about its role in lower plants. In the present study we have investigated the distribution of ubiquitin in the unicellular alga Chlamydomonas reinhardii as well as the effect of heat and light stress on its conjugation to cellular proteins. Immunoelectron microscopy shows that ubiquitin is located in the chloroplast, nucleus, cytoplasm, pyrenoid and on the plasma membrane. The location of ubiquitin within chloroplasts has not been observed previously. In immunoblots of whole cell extracts with an antibody to ubiquitin a prominent conjugate band with an apparent molecular mass of 29 kDa and a broad region of high-molecular-mass conjugates (apparent molecular mass greater than 45 kDa) were observed. Exposure of cells to a 41.5 degrees C heat shock in both the dark and light caused the disappearance of the 29-kDa conjugate and an increase in the high-molecular-mass conjugates. After step down to 25 degrees C the 29-kDa conjugate reappeared while the levels of high-molecular-mass conjugates decreased. In light, the recovery of the 29-kDa band was more rapid than in the dark. Photoinhibition alters the ubiquitin conjugation pattern similarly to heat shock, but to a lesser degree. These observations imply that, in Chlamydomonas, ubiquitin has a role in the chloroplast and in the response to heat and light stress.

The mitosis-specific monoclonal antibody MPM-2 recognizes phosphoproteins associated with the nuclear envelope in Chlamydomonas reinhardtii cells.

The monoclonal antibody MPM-2 recognizes a family of phosphorylated proteins present in mitotic cells. In a number of organisms it stains nuclei and also cytoskeletal structures which contain or organize tubulin. In mitotic Chlamydomonas reinhardtii cells MPM-2 reacts with phosphoproteins associated with the nuclear envelope (NE). Staining of the NE region appears in preprophase, reaches a maximum intensity in metaphase/anaphase and disappears rapidly in telophase. Localized hyperphosphorylation of the anterior NE region is apparent in many cells throughout mitosis. The distribution and timing of MPM-2 labeling suggests that in Chlamydomonas MPM-2 may be interacting with lamin-like phosphoproteins.

Analysis of Chlamydomonas reinhardtii histones and chromatin.

Chromatin spreads made from isolated nuclei of the unicellular green alga Chlamydomonas reinhardtii show the beaded fibers typical of eukaryotic polynucleosomes. Micrococcal nuclease digestions confirmed the presence of nucleosomes with a repeat length of 189 base pairs, essentially the same as typical mammalian cells. Basic nuclear proteins extracted from isolated nuclei or chromatin with 1 M calcium chloride and 0.3 M hydrochloric acid are resolved into seven major components by electrophoresis in the presence of sodium dodecyl sulfate (SDS). These seven components were subjected to qualitative peptide mapping with V8 protease on SDS gels for comparison with the major histone components of calf thymus. Finally, the C. reinhardtii basic nuclear proteins were fractionated by reversed phase high performance liquid chromatography and their amino acid composition determined. From these studies, we conclude that C. reinhardtii has a full complement of the five histones with properties very similar to those of both higher animals and higher plants.

Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii share features with both mitochondrial and higher plant chloroplast presequences.

Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii have been analyzed and compared with chloroplast transit peptides from higher plants and mitochondrial targeting peptides from yeast, Neurospora and higher eukaryotes. In terms of length and amino acid composition, chloroplast transit peptides from C. reinhardtii are more similar to mitochondrial targetting peptides than to chloroplast transit peptides from higher plants. They also contain the potential amphiphilic alpha-helix characteristic of mitochondrial presequences. However, in similarity with chloroplast transit peptides from higher plants, they contain a C-terminal region with the potential to form an amphiphilic beta-strand. As in higher plants, transit peptides that route proteins to the thylakoid lumen consist of an N-terminal domain similar to stroma-targeting transit peptides attached to a C-terminal apolar domain that share many characteristics with secretory signal peptides.

Purification and characterization of clathrin-coated vesicles from Chlamydomonas.

Clathrin-coated vesicles, identified by negative staining with uranyl acetate, were purified from Chlamydomonas reinhardtii. Isolated coated vesicles had diameters ranging from 70 to 140 nm (mean diameter +/- SD of 95 +/- 17 nm, n = 300). These vesicles were markedly heterogeneous in both density and surface charge, as indicated by equilibrium density sedimentation and elution from anion-exchange columns. Highly-purified coated-vesicle fractions contained 2 major polypeptides, identified as the clathrin heavy chain (185 kDa) and the clathrin light chain (40 kDa). Chlamydomonas clathrin heavy chain cross-reacts weakly with an antibody against bovine brain clathrin heavy chain. Coat stability in several buffers was compared to that of bovine brain coated vesicles. Stability was similar, except for a greater stability of Chlamydomonas coated vesicles in 0.5 M Tris at pH 7.0.

Determination of the A-T content of DNA by second-derivative ultraviolet spectroscopy.

A method for the determination of the A-T content of DNA based on second-derivative ultraviolet spectra is presented. It allows measurement in a wide range of pH values, ionic strengths, and buffer media. It is nondestructive for the sample and requires not more than 10 micrograms of DNA.

Structure and biogenesis of Chlamydomonas reinhardtii photosystem I.

The photosystem I complex of the green alga Chlamydomonas reinhardtii was isolated and fractionated into its two subcomplex components: the core complex (CC I), which contained the reaction center (P-700) and had four polypeptide subunits, and the light-harvesting complex (LHC I) which contained four polypeptides of about 22, 25, 26 and 27 kDa. The 22-kDa apoprotein was isolated as a chlorophyll a and b binding protein. In the isolated photosystem I holocomplex, about ten copies of the 22-kDa LHC I apoprotein are present for each CC I unit. The 22-kDa polypeptide as well as the other three polypeptides of this complex and the subunit II of CC I are translated on 80S cytoplasmic ribosomes, and therefore are coded in the nucleus. During the greening process of the Chlamydomonas reinhardtii y-1 mutant the 22-kDa LHC I polypeptide, which cross-reacts with polyclonal antibodies raised against the Lemna gibba 20-kDa LHC I apoprotein, accumulates in thylakoids at a late stage of their development, and about 2-3 h after the LHC II and CC I subunit II polypeptides have accumulated. Accumulation of the 22-kDa protein during greening is inhibited by cycloheximide but not by chloramphenicol.

Green algal cytochrome b6-f complexes: isolation and characterization from Dunaliella saline, Chlamydomonas reinhardtii and Scenedesmus obliquus.

Cytochrome b6-f complexes have been isolated from Chlamydomonas reinhardtii, Dunaliella saline and Scenedesmus obliquus. Each complex is essentially free of chlorophyll and carotenoids and contains cytochrome b6 and cytochrome f hemes in a 2:1 molar ratio. C. reinhardtii and S. obliquus complexes contain the Rieske iron-sulfur protein (present in approx 1:1 molar ratio to cytochrome f) and each catalyzes a DBMIB- and DNP-INT-sensitive electron transfer from duroquinol to spinach plastocyanin. Immunological assays using antibodies to the peptides from the spinach cytochrome complex show varying cross-reactivity patterns except for the complete absence of binding to the Rieske proteins in any of the three complexes, suggesting little structural similarity between the Rieske proteins of algae with those from higher plants. One complex (D. salina) has been uniformly labeled by growth in NaH14CO3 to determine stoichiometries of constituent polypeptide subunits. Results from these studies indicate that all functionally active cytochrome b6-f complexes contain four subunits which occur in equimolar amounts.

Purification and characterization of a basal body-associated Ca2+-binding protein.

Isolated basal body complexes from the unicellular alga, Chlamydomonas reinhardtii were found to contain a low molecular mass acidic polypeptide, distinct from calmodulin, but with biochemical features in common with members of the calmodulin family of calcium-binding proteins. These common characteristics included a relative low molecular mass of 20 kD, an experimentally determined acidic pI of 5.3, an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of added calcium, and a calcium-dependent binding to the hydrophobic ligand phenyl-Sepharose which allowed its purification by affinity chromatography. The relatedness of the basal body-associated 20-kD calcium-binding protein (CaBP) to calmodulin was confirmed by amino acid compositional analysis and partial peptide sequencing of the isolated protein. A rabbit antibody specific for the 20-kD CaBP was raised and used to determine by indirect immunofluorescence the cellular localization of the protein in Chlamydomonas cells. In interphase cells the antibody stained intensely the region between the paired basal bodies, two fibers extending between the basal bodies and the underlying nucleus, and an array of longitudinal filaments surrounding the nucleus. The two basal body-nuclear connecting fibers were identified in thin-section electron micrographs to be narrow striated fiber roots. In mitotic cells the 20-kD CaBP was specifically associated with the poles of the mitotic spindle at the sites of the duplicated basal body complexes.

Molecular cloning of cDNA for caltractin, a basal body-associated Ca2+-binding protein: homology in its protein sequence with calmodulin and the yeast CDC31 gene product.

An extended synthetic oligonucleotide (59-mer) was used to isolate a Chlamydomonas cDNA containing the entire coding region for a novel basal body-associated 20-kD calcium-binding protein (CaBP). DNA and RNA blot analysis indicate that the 20-kD CaBP is encoded by a single copy gene from which is derived an approximately 1.1-kb-long transcript. The deduced amino acid sequence for the protein shows a linear relatedness with calmodulin from Chlamydomonas and other organisms (45-48% identity). The primary protein sequence of the 20-kD CaBP and its predicted secondary structure suggests that the protein is likely to contain four homologous calcium-binding domains that conform to the helix-loop-helix (or EF hand) structure found in calmodulin and related calcium-modulated proteins. The major difference between the protein and calmodulin is an amino-terminal domain of 21 amino acids present on the 20-kD CaBP. In addition to its relatedness to calmodulin, the Chlamydomonas 20-kD CaBP shows a strong sequence identity (50%) with the yeast Saccharomyces cerevisiae CDC31 gene product required for spindle pole body duplication. The association of these sequence-related calcium-binding proteins to microtubule-organizing centers of divergent structure suggests a potential conserved function for the proteins.

Purification, properties and complete amino acid sequence of the ferredoxin from a green alga, Chlamydomonas reinhardtii.

The ferredoxin was purified from the green alga, Chlamydomonas reinhardtii. The protein showed typical absorption and circular dichroism spectra of a [2Fe-2S] ferredoxin. When compared with spinach ferredoxin, the C. reinhardtii protein was less effective in the catalysis of NADP+ photoreduction, but its activity was higher in the light activation of C. reinhardtii malate dehydrogenase (NADP). The complete amino acid sequence was determined by automated Edman degradation of the whole protein and of peptides obtained by trypsin and chymotrypsin digestions and by CNBr cleavage. The protein consists of 94 residues, with Tyr at both NH2 and COOH termini. The positions of the four cysteines binding the two iron atoms are similar to those found in other [2Fe-2S] ferredoxins. The primary structure of C. reinhardtii ferredoxin showed a great homology (about 80%) with ferredoxins from two other green algae.

Isolation of a sixth dynein subunit adenosine triphosphatase of Chlamydomonas axonemes.

This study of the axoneme led to the identification of a previously unknown adenosine triphosphatase (ATPase), which is likely a major component of inner dynein arms. The ATPase was isolated from a soluble fraction of axonemes obtained from pf 28, a Chlamydomonas mutant lacking the outer dynein arms. The activity hydrolyzed up to 2.3 mumol of ATP.min-1.mg-1 of protein (at pH 7.2, in the presence of both Ca++ and Mg++), had a sedimentation coefficient of 11S in sucrose gradient, and cosedimented with four polypeptides of apparent molecular weight 325,000, 315,000 140,000, and 42,000. Several arguments indicate that the new ATPase is a component of the inner dynein arms. Three or four polypeptides cosedimenting with the activity belong to a group of axonemal components that are deficient in the axonemes of pf 23 and pf 30, two mutants that display different levels of inner dynein arm deficiency. The 42,000 component is axonemal actin, a subunit of two other inner dynein ATPases. The two polypeptides of molecular weight greater than 300,000 have electrophoretic mobility similar to that of high molecular weight components of outer and inner dynein arms. In spite of some similarities each ATPase isolated from inner or outer arms is composed of a different set of polypeptides. Different ATPases may be required for the modulation of localized sliding of adjacent outer double microtubules in the axoneme.

Use of carbohydrate probes in conjunction with fluorescence-activated cell sorting to select mutant cell lines of Chlamydomonas with defects in cell surface glycoproteins.

Two carbohydrate-binding probes (the lectin concanavalin A and the anti-carbohydrate monoclonal antibody FMG-1) have been utilized in conjunction with fluorescence-activated cell sorting to select cell lines of Chlamydomonas reinhardtii that contain defects in cell surface-exposed glycoproteins. Two very different selection strategies (sorting cells with the lowest binding for the FMG-1 monoclonal antibody or the highest binding of concanavalin A) yield a class of mutant cells that exhibit a total lack of binding of the monoclonal antibody to cell wall and plasma membrane glycoproteins along with an increased affinity for concanavalin A. Detailed characterization of one such mutant cell line, designated L-23, is provided. The subtle glycosylation defect exhibited by this cell line does not alter the ability of the affected glycoproteins to be targeted to the flagellar membrane and does not affect the expression of flagellar surface motility, a phenomenon that appears to involve the major concanavalin A-binding glycoprotein of the flagellar membrane. This approach has general applicability for dissecting the role of carbohydrate epitopes in the targeting and function of any cell surface glycoprotein for which suitable carbohydrate probes are available.