ABSTRACTWhen the unicellular green soil algaChlamydomonas reinhardtiiis deprived of nitrogen after entering stationary phase in liquid culture, the cells produce abundant cytoplasmic lipid bodies (LBs), as well as abundant starch, via a pathway that accompanies a regulated autophagy program. After 48 h of N starvation in the presence of acetate, the wild-type LB content has increased 15-fold. When starch biosynthesis is blocked in thesta6mutant, the LB content increases 30-fold, demonstrating that genetic manipulation can enhance LB production. The use of cell wall-less strains permitted development of a rapid “popped-cell” microscopic assay to quantitate the LB content per cell and permitted gentle cell breakage and LB isolation. The highly purified LBs contain 90% triacylglycerol (TAG) and 10% free fatty acids (FFA). The fatty acids associated with the TAGs are ∼50% saturated (C16and C18) fatty acids and ∼50% unsaturated fatty acids, half of which are in the form of oleic acid (C18:1). The FFA are ∼50% C16and ∼50% C18. The LB-derived TAG yield from a liter ofsta6cells at 107cells/ml after starvation for 48 h is calculated to approach 400 mg. The LB fraction also contains low levels of charged glycerolipids, with the same profile as whole-cell charged glycerolipids, that presumably form LB membranes; chloroplast-specific neutral glycerolipids (galactolipids) are absent. Very low levels of protein are also present, but all matrix-assisted laser desorption ionization-identified species are apparent contaminants. Nitrogen stress-induced LB production inC. reinhardtiihas the hallmarks of a discrete pathway that should be amenable to additional genetic and culture condition manipulation.

The evolution of anisogamy/oogamy in the colonial Volvocales might have occurred in an ancestral isogamous colonial organism like Gonium pectorale. The unicellular, close relative Chlamydomonas reinhardtii has a mating-type (MT) locus harboring several mating-type-specific genes, including one involved in mating-type determination and another involved in the function of the tubular mating structure in only one of the two isogametes. In this study, as the first step in identifying the G. pectorale MT locus, we isolated from G. pectorale the ortholog of the C. reinhardtii mating-type-determining minus-dominance (CrMID) gene, which is localized only in the MT− locus. 3′- and 5′-RACE RT–PCR using degenerate primers identified a CrMID-orthologous 164-amino-acid coding gene (GpMID) containing a leucine-zipper RWP-RK domain near the C-terminal, as is the case with CrMID. Genomic Southern blot analysis showed that GpMID was coded only in the minus strain of G. pectorale. RT–PCR revealed that GpMID expression increased during nitrogen starvation. Analysis of F1 progeny suggested that GpMID and isopropylmalate dehydratase LEU1S are tightly linked, suggesting that they are harbored in a chromosomal region under recombinational suppression that is comparable to the C. reinhardtii MT locus. However, two other genes present in the C. reinhardtii MT locus are not linked to the G. pectorale LEU1S/MID, suggesting that the gene content of the volvocalean MT loci is not static over time. Inheritance of chloroplast and mitochondria genomes in G. pectorale is uniparental from the plus and minus parents, respectively, as is also the case in C. reinhardtii.

Protein diversification is commonly driven by single amino acid changes at random positions followed by selection, but, in some cases, the structure of the gene itself favors the occurrence of particular kinds of mutations. Genes encoding hydroxyproline-rich glycoproteins (HRGPs) in green organisms, key protein constituents of the cell wall, carry short-repeat modules that are posited to specify proline hydroxylation and/or glycosylation events. We show here, in a comparison of two closely related Chlamydomonas species-Chlamydomonas reinhardtii (CC-621) and Chlamydomonas incerta (CC-1870/3871)-that these modules are prone to misalignment and hence to both insertion/deletion and endoduplication events, and that the dynamics of the rearrangements are constrained by purifying selection on the repeat patterns themselves, considered either as helical or as longitudinal face modules. We suggest that such dynamics may contribute to evolutionary diversification in cell wall architecture and physiology. Two of the HRGP genes analyzed (SAG1 and SAD1) encode the mating-type plus and minus sexual agglutinins, displayed only by gametes, and we document that these have undergone far more extensive divergence than two HRGP genes (GP1 and VSP3) that encode cell wall components-an example of the rapid evolution that characterizes sex-related proteins in numerous lineages. Strikingly, the central regions of the agglutinins of both mating types have diverged completely, by selective endoduplication of repeated motifs, since the two species last shared a common ancestor, suggesting that these events may have participated in the speciation process.

Linear hydroxyproline-bound O-glycans of the unicellular green alga Chlamydomonas reinhardtii were isolated from outer cell wall glycoproteins and their structure elucidated by chemical and spectroscopic methods. They consist exclusively of arabinose and galactose, the latter in the furanose form, unusual for plants. The first two arabinoses linked to hydroxyproline have the same anomeric configuration and linkage to each other as do the arabinosides isolated from land plants, suggesting that these two steps of hydroxyproline-O-glycosylation have been conserved during evolution. In a concomitant analysis of Hyp-bound O-tri- and tetraarabinans isolated from Arabidopsis thaliana, we were able to unambiguously identify the natural trans-4-l-Hyp-bound O-glycans and the artificially produced d-allo forms and to identify diagnostic signals in NMR spectra of these compounds for the first time.

AbstractGametes of the unicellular green alga Chlamydomonas reinhardtii undergo sexual adhesion via enormous chimeric Hyp-rich glycoproteins (HRGPs), the plus and minus sexual agglutinins, that are displayed on their flagellar membrane surfaces. We have previously purified the agglutinins and analyzed their structural organization using electron microscopy. We report here the cloning and sequencing of the Sag1 and Sad1 genes that encode the two agglutinins and relate their derived amino acid sequences and predicted secondary structure to the morphology of the purified proteins. Both agglutinin proteins are organized into three distinct domains: a head, a shaft in a polyproline II configuration, and an N-terminal domain. The plus and minus heads are related in overall organization but poorly conserved in sequence except for two regions of predicted hydrophobic α-helix. The shafts contain numerous repeats of the PPSPX motif previously identified in Gp1, a cell wall HRGP. We propose that the head domains engage in autolectin associations with the distal termini of their own shafts and suggest ways that adhesion may involve head–head interactions, exolectin interactions between the heads and shafts of opposite type, and antiparallel shaft–shaft interactions mediated by carbohydrates displayed in polyproline II configurations.

Gametes of the unicellular green alga Chlamydomonas reinhardtii undergo sexual adhesion via enormous chimeric Hyp-rich glycoproteins (HRGPs), the plus and minus sexual agglutinins, that are displayed on their flagellar membrane surfaces. We have previously purified the agglutinins and analyzed their structural organization using electron microscopy. We report here the cloning and sequencing of the Sag1 and Sad1 genes that encode the two agglutinins and relate their derived amino acid sequences and predicted secondary structure to the morphology of the purified proteins. Both agglutinin proteins are organized into three distinct domains: a head, a shaft in a polyproline II configuration, and an N-terminal domain. The plus and minus heads are related in overall organization but poorly conserved in sequence except for two regions of predicted hydrophobic a-helix. The shafts contain numerous repeats of the PPSPX motif previously identified in Gp1, a cell wall HRGP. We propose that the head domains engage in autolectin associations with the distal termini of their own shafts and suggest ways that adhesion may involve head-head interactions, exolectin interactions between the heads and shafts of opposite type, and antiparallel shaft-shaft interactions mediated by carbohydrates displayed in polyproline II configurations.

In Saccharomyces cerevisiae four transporters, Tpo1p–Tpo4p, all members of the major facilitator superfamily, have been shown to confer resistance to polyamines. It was suggested that they act by pumping their respective substrate into the lumen of the vacuole depending on the proton gradient generated by the V-ATPase. Using sucrose gradient ultracentrifugation we found that an hemagglutinin (HA)-tagged Tpo1p as well as its HA-tagged Tpo2p–4p homologues co-localize with plasma membrane markers. Because the HA-tagged Tpo1p carrier protein proved to be functional in conferring resistance to polyamines in TPO1 knockouts, a function of Tpo1p in transport of polyamines across the plasma membrane seemed to be likely. The polyamine transport activity of wild type cells was compared with the respective activity of a TPO1 knockout strain. The results obtained strongly suggest that Tpo1p is a plasma membrane-bound exporter, involved in the detoxification of excess spermidine in yeast. When studying polyamine transport of wild type cells, we furthermore found that S. cerevisiae is excreting putrescine during the fermentative growth phase.

X-ray crystallography and NMR contain complementary information for the structural characterization of biological macromolecules. X-ray diffraction is primarily sensitive to the overall shape of the molecule, whereas NMR is mostly sensitive to the atomic detail. Their combination can therefore provide a stronger justification for the resulting structure. For their combination we have recently proposed REFMAC-NMR, which relies on primary data from both techniques for joint refinement. This possibility raises the compelling question of how far the complementarity can be extended. In this paper, we describe an integrative approach to the refinement with NMR data of four X-ray structures of hen-egg-white lysozyme, solved at atomic resolution in four different crystal forms, and we demonstrate that the outcome critically depends on the crystal form itself, reflecting the sensitivity of NMR to fine details.

The Arabidopsis thalianachlorophyll a/b binding protein underexpressed (cue1) mutant, which has been isolated in a screen for chlorophyll a/b binding protein (CAB) underexpressors, exhibits a reticulate leaf phenotype combined with delayed chloroplast development and aberrant shape of the palisade parenchyma cells. The affected gene in cue1 is a phosphoenolpyruvate (PEP)/phosphate translocator (PPT) of the plastid inner envelope membrane. The proposed function of the PPT in C3-plants is the import of PEP into the stroma as one of the substrates for the shikimate pathway, from which aromatic amino acids and a variety of secondary plant products derive. The mutant phenotype could be: (i) complemented by constitutive overexpression of a heterologous PPT from cauliflower; and (ii) rescued by overexpression of a C4-type pyruvate,orthophosphate dikinase (PPDK). The latter approach indicates that PEP deficiency within plastids triggers developmental constraints in cue1. The impact of the mutation on aspects of primary and secondary metabolism was assessed in cue1 as well as in the individual transformant lines. The majority of the data obtained in this and an accompanying paper suggest that the mutant phenotype is not simply caused by a general restriction of the shikimate pathway because of a defect in a PPT.

Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide bridged, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF-pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O-glycosylated proteins up to 150 kDa after SDS-PAGE separation.

Hydroxyproline-rich glycoproteins (HRGPs) are the major proteinaceous components of higher plant walls and the predominant components of the cell wall of the green alga Chlamydomonas reinhardtii. The GP1 protein, an HRGP of the C. reinhardtii wall, is shown to adopt a polyproline II helical configuration and to carry a complex array of arabinogalactoside residues, many branched, which are necessary to stabilize the helical conformation. The deduced GP1 amino acid sequence displays two Ser-Pro-rich domains, one with a repeating (SP)(x)() motif and the other with a repeating (PPSPX)(x)() motif. A second cloned gene a2 also carries the PPSPX repeat, defining a novel gene family in this lineage. The SP-repeat domains of GP1 form a 100-nm shaft with a flexible kink 28 nm from the head. The gp1 gene encodes a PPPPPRPPFPANTPM sequence at the calculated kink position, generating the proposal that this insert interrupts the PPII helix, with the resultant kink exposing amino acids necessary for GP1 to bind to partner molecules. It is proposed that similar kinks in the higher plant HRGPs called extensins may play a comparable role in wall assembly.

Of all algae, those in the division chlorophyta (green algae) display the closest relationship to the vascular plants. Chlorophytes harbor a chloroplast that is considered to have originated from a single endosymbiotic event and contain the same types of photosynthetic pigments as land plants. At

The cell wall of Chlamydomonas reinhardtii zygotes, which forms rapidly after the fusion of wall‐free gametes, provides a tractable system for studying the properties and assembly of hydroxyproline‐rich glycoproteins, the major proteinaceous components of green algal and plant cell walls. We report the cloning of the zsp2 gene and the analysis of its ZSP‐2 product, a 58.9 kDa polypeptide that is synthesized exclusively by zygotes. The protein contains two (SP)x repeats, establishing it as a member of the cell wall hydroxyproline‐rich glycoproteins family. It also contains a 4‐fold iteration of an amino acid sequence centered around cysteine residues, a configuration found in both plant and animal lectins. Furthermore, we report four observations on pellicle composition and production. First, cell‐free preparations of the pellicle matrix are rich in hydroxyproline, arabinose, and galactose and contain bundles of very long fibrils. Second, glutathione blocks pellicle formation and results in the accumulation of long fibrils in the growth medium. Third, antibody to ZSP‐2 also blocks pellicle formation. Fourth, ZSP‐2 immunolocalizes to the boundary between the outer layers of the wall proper and the pellicle matrix. These observations are consistent with the possibility that the Cys‐rich (glutathione‐sensitive) lectin‐like domains of ZSP‐2 may bind to sugar residues on the long fibrils and anchor them to the cell wall, thereby initiating and maintaining pellicle formation.

Mass spectrometric techniques are presented which allow one to analyze the sugar part bound to hydroxyproline in hydroxyproline-rich glycoproteins. The hydroxyproline (Hyp) glycans obtained by alkaline hydrolysis give abundant [M + Na](+) ions by electrospray ionization which after collision-induced dissociation (CID) yield inter alia [Hyp - H + Na](+). In mixtures a parent ion scan of this species will indicate the various molecular species which can then be analyzed by MS(n) after CID in an ion trap, where successive losses of the sugar units are observed. Methylation techniques allow one to distinguish between linear and branched isomeric structures.

The addition of primary amines to the growth medium of the unicellular green alga Chlamydomonas reinhardtii disrupts cell wall assembly in both vegetative and zygotic cells. Primary amines are competitive inhibitors of the protein-cross-linking activity of transglutaminases. Two independent assays for transglutaminase confirmed a burst of extracellular activity during the early stages of cell wall formation in both vegetative cells and zygotes. When non-inhibiting levels of a radioactive primary amine ((14)C-putrescine) were added to the growth medium, both cell types were labeled in a reaction catalyzed by extracellular transglutaminase. The radioactive label was found specifically in the cell wall proteins of both cell types, and acid hydrolysis of the labeled material released unmodified (14)C-putrescine. Western blots of the proteins secreted at the times of maximal transglutaminase activity in both cell types revealed a single highly cross-reactive 72-kD band when screened with antibodies to guinea pig tissue transglutaminase. Furthermore, the proteins immunoprecipitated by this antiserum in vivo exhibited transglutaminase activity. We propose that this transglutaminase is responsible for an early cell wall protein cross-linking event that temporally precedes the oxidative cross-linking mediated by extracellular peroxidases.

Enzymatic removal of the cell wall induces vegetative Chlamydomonas reinhardtii cells to transcribe wall genes and synthesize new hydroxyproline-rich glycoproteins (HRGPs) related to the extensins found in higher plant cell walls. A cDNA expression library made from such induced cells was screened with antibodies to an oligopeptide containing the (SP)x repetitive domains found in Chlamydomonas wall proteins. One of the selected cDNAs encodes an (SP)x-rich polypeptide that also displays a repeated YGG motif. Ascorbate, a peroxidase inhibitor, and tyrosine derivatives were shown to inhibit insolubilization of both the vegetative and zygotic cell walls of Chlamydomonas, suggesting that oxidative cross-linking of tyrosines is occurring. Moreover, insolubilization of both walls was concomitant with a burst in H2O2 production and in extracellular peroxidase activity. Finally, both isodityrosine and dityrosine were found in hydrolysates of the insolubilized vegetative wall layer. We propose that the formation of tyrosine cross-links is essential to Chlamydomonas HRGP insolubilization.

AbstractGlykoproteine sind, wie der Name sagt, mit Kohlenhydraten mehr order weniger stark kovalent modifizierte Proteine, wobei der Kohlenhydratanteil zwischen einem und 80 Prozent liegt. Glykoproteine kommen in allen Lebewesen (Pro‐ und Eukaryonten) vor und haben die unterschiedlichsten Funktionen, die vielfach auf spezifische Wechselwirkungen zwischen den zuckerhaltigen Molekülanteilen zurückzuführen sind. Pflanzliche Glykoproteine sind erst in jüngerer Zeit „interessant„ geworden, als man erkannte, daß sie für viele agronomische Vorkommnisse verantwortlich sind, zum Beispiel für die Bindung und Einbindung von Symbionten bei der Knöllchenbildung der Leguminosen, bei der Infektion von Pflanzen mit Viren und Pilzen, bei den Vorgängen der Bestäubung, Befruchtung und Selbststerilität. In der Tat haben Pflanzen eine Art Immunsystem auf diesen Prinzipien entwickelt, das bereit steht, die ökologischen Wechselbeziehungen zu stabilisieren oder zu verteidigen, und damit für die ökonomischen Wechselbeziehungen zwischen Mensch und Primärnahrung wesentlich ist.

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