Chlamydomonas Mutants Research Articles

Dimeric Novel HSP40 Is Incorporated into the Radial Spoke Complex during the Assembly Process in Flagella

The radial spoke is a stable structural complex in the 9 + 2 axoneme for the control of flagellar motility. However, the spokes in Chlamydomonas mutant pf24 are heterogeneous and unstable, whereas several spoke proteins are reduced differentially. To elucidate the defective mechanism, we clone RSP16, a prominent spoke protein diminished in pf24 axonemes. Unexpectedly, RSP16 is a novel HSP40 member of the DnaJ superfamily that assists chaperones in various protein-folding-related processes. Importantly, RSP16 is uniquely excluded from the 12S spoke precursor complex that is packaged in the cell body and transported toward the flagellar tip to be converted into mature 20S axonemal spokes. Rather, RSP16, transported separately, joins the precursor complex in flagella. Furthermore, RSP16 molecules in vitro and in flagella form homodimers, a characteristic required for the cochaperone activity of HSP40. We postulate that the spoke HSP40 operates as a cochaperone to assist chaperone machinery at the flagellar tip to actively convert the smaller spoke precursor and itself into the mature stable complex; failure of the interaction between the spoke HSP40 and its target polypeptide results in heterogeneous unstable radial spokes in pf24.

A Tektin Homologue Is Decreased inChlamydomonasMutants Lacking an Axonemal Inner-Arm Dynein

In ciliary and flagellar axonemes, various discrete structures such as inner and outer dynein arms are regularly arranged on the outer doublet microtubules. Little is known about the basis for their regular arrangement. In this study, proteins involved in the attachment of inner-arm dyneins were searched by a microtubule overlay assay on Chlamydomonas mutant axonemes. A 58-kDa protein (p58) was found approximately 80% diminished in the mutants ida6 and pf3, both lacking one (species e) of the seven inner-arm species (a-g). Analysis of its cDNA indicated that p58 is homologous to tektin, a protein that was originally found in sea urchin and thought to be crucial for the longitudinal periodicity of the doublet microtubule. Unlike sea urchin tektin, which is a component of protofilament ribbons that occur after Sarkosyl treatment of axonemes, p58 was not contained in similar Sarkosyl-resistant ribbons from Chlamydomonas axonemes. Immunofluorescence microscopy showed that p58 was localized uniformly along the axoneme and on the basal body. The p58 signal was reduced in ida6 and pf3. These results suggest that a reduced amount of p58 is sufficient for the production of outer doublets, whereas an additional amount of it is involved in inner-arm dynein attachment.

Starch granule initiation is controlled by a heteromultimeric isoamylase in potato tubers.

Starch granule initiation is not understood, but recent evidence implicates a starch debranching enzyme, isoamylase, in the control of this process. Potato tubers contain isoamylase activity attributable to a heteromultimeric protein containing Stisa1 and Stisa2, the products of two of the three isoamylase genes of potato. To discover whether this enzyme is involved in starch granule initiation, activity was reduced by expression of antisense RNA for Stisa1 or Stisa2. Transgenic tubers accumulated a small amount of a soluble glucan, similar in structure to the phytoglycogen of cereal, Arabidopsis, and Chlamydomonas mutants lacking isoamylase. The major effect, however, was on the number of starch granules. Transgenic tubers accumulated large numbers of tiny granules not seen in normal tubers. These data indicate that the heteromultimeric isoamylase functions during starch synthesis to suppress the initiation of glucan molecules in the plastid stroma that would otherwise crystallize to nucleate new starch granules.

Evidence That the PsbK Polypeptide Is Associated with the Photosystem II Core Antenna Complex CP43

PsbK is encoded by the chloroplast psbK gene and is one of the small polypeptides of photosystem II (PSII). This polypeptide is required for accumulation of the PSII complex. In the present study, we generated an antibody against recombinant mature PsbK of Chlamydomonas and used it in Western blots to localize PsbK in the PSII core complex. PsbK was found in the thylakoid membranes, and purification of the PSII core complex from detergent-solubilized thylakoid membranes showed that PsbK is tightly associated with the PSII core complex. We used potassium thiocyanate to separate PSII into subcore complexes, including the D1/D2/cytochrome b559 reaction center complex, CP47, and CP43, and we found that PsbK co-purifies with one of the core antenna complexes, CP43, during ion exchange chromatography. Subsequent gel filtration chromatography of the purified CP43 confirmed that PsbK is tightly associated with CP43. Steady-state levels of PsbK were also determined in Chlamydomonas mutants expressing various levels of PSII. Quantitative Western blotting revealed that the levels of PsbK in these mutants are approximately equal to those of CP43, suggesting that PsbK is stable only when associated with CP43 in the chloroplast. Together, our results indicate that PsbK is an integral part of the PSII complex and may participate in the assembly and stability of the PSII complex.

Decrease in the efficiency of the electron donation to tyrosine Z of photosystem II in an SQDG-deficient mutant of Chlamydomonas

Photosystem (PS) II activity of a sulfoquinovosyl diacylglycerol (SQDG)-deficient mutant ( hf-2) of Chlamydomonas was partially decreased compared with that of wild-type. The susceptibility to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was also modified in the mutant. Photometric measurements in the isolated thylakoid membranes of hf-2 revealed that the lowered activity in the mutant was derived from a decrease in the efficiency of the electron donation from water to tyrosine Z, not from the efficiency of the electron transport from Q A to Q B. This result was confirmed by the decay kinetics of chlorophyll fluorescence determined in vivo. We conclude that SQDG contributes to maintaining the conformation of PSII complexes, particularly that of D1 polypeptides, which are necessary for maximum activities in Chlamydomonas.

Structural analysis of three-spin systems of photosystem II by PELDOR

The pulsed electron electron double resonance (PELDOR) pulse sequence is applied to a three-spin system consisting of three radicals (YD·, YZ· and QA−) generated in spinach PS II. The distance between YZ and QA has been determined to be 3.4 nm with the previously derived distances of the other radical pairs, 2.9 nm for YD·-YZ· and 3.9 nm for YD·-QA−. This distance has been derived from the YZ·-QA− radical pair trapped in YD-less mutants ofChlamydomonas reinhardtii. Furthermore the method was applied to the YD·-QA−-ChlZ+ system to find the unknown distance between QA and ChlZ. The derived distance was 3.4 nm. A triangular configuration was found in the membrane system that gives the relative positions of the electron transfer components.

A Novel MAP Kinase Regulates Flagellar Length in Chlamydomonas

Little is known about the molecular basis of organelle size control in eukaryotes. Cells of the biflagellate alga Chlamydomonas reinhardtii actively maintain their flagella at a precise length. Chlamydomonas mutants that lose control of flagellar length have been isolated and used to demonstrate that a dynamic process keeps flagella at an appropriate length [1, 2]. To date, none of the proteins required for flagellar length control have been identified in any eukaryotic organism. Here, we show that a novel MAP kinase is crucial to enforcing wild-type flagellar length in C. reinhardtii. Null mutants of LF4[2], a gene encoding a protein with extensive amino acid sequence identity to a mammalian MAP kinase of unknown function, MOK [3], are unable to regulate the length of their flagella. The LF4 protein (LF4p) is localized to the flagella, and in vitro enzyme assays confirm that the protein is a MAP kinase. The long-flagella phenotype of lf4 cells is rescued by transformation with the cloned LF4 gene. The demonstration that a novel MAP kinase helps enforce flagellar length control indicates that a previously unidentified signal transduction pathway controls organelle size in C. reinhardtii.

DC3, the 21-kDa subunit of the outer dynein arm-docking complex (ODA-DC), is a novel EF-hand protein important for assembly of both the outer arm and the ODA-DC.

The outer dynein arm-docking complex (ODA-DC) is a microtubule-associated structure that targets the outer dynein arm to its binding site on the flagellar axoneme (Takada et al. 2002. Mol. Biol. Cell 13, 1015-1029). The ODA-DC of Chlamydomonas contains three proteins, referred to as DC1, DC2, and DC3. We here report the isolation and sequencing of genomic and full-length cDNA clones encoding DC3. The sequence predicts a 21,341 Da protein with four EF-hands that is a member of the CTER (calmodulin, troponin C, essential and regulatory myosin light chains) group and is most closely related to a predicted protein from Plasmodium. The DC3 gene, termed ODA14, is intronless. Chlamydomonas mutants that lack DC3 exhibit slow, jerky swimming because of loss of some but not all outer dynein arms. Some outer doublet microtubules without arms had a "partial" docking complex, indicating that DC1 and DC2 can assemble in the absence of DC3. In contrast, DC3 cannot assemble in the absence of DC1 or DC2. Transformation of a DC3-deletion strain with the wild-type DC3 gene rescued both the motility phenotype and the structural defect, whereas a mutated DC3 gene was incompetent to rescue. The results indicate that DC3 is important for both outer arm and ODA-DC assembly.

Centrioles get a helping hand from centrin

Although the precision of centriole duplication has aroused interest for over a century, its molecular mechanism remains almost entirely unknown. What proteins are required for a pre-existing centriole to induce assembly of a new centriole? One strong candidate is centrin, an EF hand-containing calcium binding protein first identified biochemically in green algae. Analysis of centrin mutants in the haploid unicellular alga Chlamydomonas (also known as ‘green yeast’ because of its powerful genetics) suggests that centrin is required for proper centriole segregation, and that defects in centrin lead to defects in centriole duplication. However, these studies leave several important questions about centrin function unanswered. First, centriole duplication still occurs in the Chlamydomonas centrin mutant, albeit to a reduced extent. Is this because the mutation, which was not a null, retains some residual centrin function, or because centrin is not strictly required for centriole duplication? Second, although the centrioles of Chlamydomonas are virtually identical to those of animal cells, is it possible that centrin might play a different role in animals?

Centrioles get a helping hand from centrin

Although the precision of centriole duplication has aroused interest for over a century, its molecular mechanism remains almost entirely unknown. What proteins are required for a pre-existing centriole to induce assembly of a new centriole? One strong candidate is centrin, an EF hand-containing calcium binding protein first identified biochemically in green algae. Analysis of centrin mutants in the haploid unicellular alga Chlamydomonas (also known as'green yeast' because of its powerful genetics) suggests that centrin is required for proper centriole segregation, and that defects in centrin lead to defects in centriole duplication. However, these studies leave several important questions about centrin function unanswered. First, centriole duplication still occurs in the Chlamydomonas centrin mutant, albeit to a reduced extent. Is this because the mutation, which was not a null, retains some residual centrin function, or because centrin is not strictly required for centriole duplication? Second, although the centrioles of Chlamydomonas are virtually identical to those of animal cells, is it possible that centrin might play a different role in animals?To resolve these questions, Jeffrey Salisbury [1xCentrin-2 is required for centriole duplication in mammalian cells. Salisbury, J.L. et al. Curr. Biol. 2002; 12: 1287–1292Abstract | Full Text | Full Text PDF | PubMed | Scopus (202)See all References[1] (who, by the way, was the first to discover centrin in algae) and colleagues have used RNA interference (RNAi) to reduce the levels of centrin-2 (the centriole-specific centrin isoform) in human tissue culture cells. RNAi of centrin-2 in HeLa cells leads to progressive loss of centrioles, consistent with a complete block in centriole replication. This result confirms that centrin is involved in centriole duplication in animal cells, which was suggested in previous work with algae. Moreover, these results show that a significant reduction in centrin levels can, in fact, cause a complete block in centriole duplication. This implies that the requirement for centrin is absolute.Interestingly, although centriole duplication is blocked, cell division continues and bipolar spindles are still observed, even when centrioles are absent. However, these acentriolar spindles are abnormal, with broad, non-focused poles. In addition, multinucleate cells are observed. These results are consistent with a function for centrioles in both spindle assembly and cytokinesis.These results indicate that centrin plays a conserved role in centriole duplication in eukaryotes ranging from algae to animals. The yeast homolog of centrin, CDC31, is a component of the half-bridge structure involved in spindle-pole body duplication. Thus, these results suggest that centriole duplication might involve a structure similar to the half-bridge in yeast.

Regulation of flagellar dynein by calcium and a role for an axonemal calmodulin and calmodulin-dependent kinase.

Ciliary and flagellar motility is regulated by changes in intraflagellar calcium. However, the molecular mechanism by which calcium controls motility is unknown. We tested the hypothesis that calcium regulates motility by controlling dynein-driven microtubule sliding and that the central pair and radial spokes are involved in this regulation. We isolated axonemes from Chlamydomonas mutants and measured microtubule sliding velocity in buffers containing 1 mM ATP and various concentrations of calcium. In buffers with pCa > 8, microtubule sliding velocity in axonemes lacking the central apparatus (pf18 and pf15) was reduced compared with that of wild-type axonemes. In contrast, at pCa4, dynein activity in pf18 and pf15 axonemes was restored to wild-type level. The calcium-induced increase in dynein activity in pf18 axonemes was inhibited by antagonists of calmodulin and calmodulin-dependent kinase II. Axonemes lacking the C1 central tubule (pf16) or lacking radial spoke components (pf14 and pf17) do not exhibit calcium-induced increase in dynein activity in pCa4 buffer. We conclude that calcium regulation of flagellar motility involves regulation of dynein-driven microtubule sliding, that calmodulin and calmodulin-dependent kinase II may mediate the calcium signal, and that the central apparatus and radial spokes are key components of the calcium signaling pathway.

Regulation of flagellar dynein by the axonemal central apparatus

Numerous studies indicate that the central apparatus, radial spokes, and dynein regulatory complex form a signaling pathway that regulates dynein activity in eukaryotic flagella. This regulation involves the action of several kinases and phosphatases anchored to the axoneme. To further investigate the role of the central apparatus in this signaling pathway, we have taken advantage of a microtubule-sliding assay to assess dynein activity in central apparatus defective mutants of Chlamydomonas. Axonemes isolated from both pf18 and pf15 (lacking the entire central apparatus) and from pf16 (lacking the C1 central microtubule) have reduced microtubule-sliding velocity compared with wild-type axonemes. Based on functional analyses of axonemes isolated from radial spokeless mutants, we hypothesized that inhibitors of casein kinase 1 (CK1) and cAMP dependent protein kinase (PKA) would rescue dynein activity and increase microtubule-sliding velocity in central pairless mutants. Treatment of axonemes isolated from both pf18 and pf16 with DRB, a CK1 inhibitor, but not with PKI, a PKA inhibitor, restored dynein activity to wild-type levels. The DRB-induced increase in dynein-driven microtubule sliding was inhibited if axonemes were first incubated with the phosphatase inhibitor, microcystin. Inhibiting CK1 in pf15 axonemes, which lack the central pair as well as PP2A [Yang et al., 2000: J. Cell Sci. 113:91-102], did not increase microtubule-sliding velocity. These data are consistent with a model in which the central apparatus, and specifically the C1 microtubule, regulate dynein through interactions with the radial spokes that ultimately alter the activity of CK1 and PP2A. These data are also consistent with localization of axonemal CK1 and PP2A near the dynein arms.

Functional diversity of axonemal dyneins as studied in Chlamydomonas mutants

Cilia and flagella of most organisms are equipped with two kinds of motor protein complex, the inner and outer dynein arms. The two arms were previously thought to be similar to each other, but recent studies using Chlamydomonas mutants indicate that they differ significantly in subunit structure and arrangement within the axoneme. For example, whereas the outer dynein arm exists as a single protein complex containing three heavy chains, the inner dynein arm comprises seven different subspecies each containing one or two discrete heavy chains. Furthermore, the two kinds of arms appear to differ in function also. Most strikingly, our studies suggest that inner-arm dynein, but not outer-arm dynein, is under the control of the central pair microtubules and radial spokes. The axoneme thus appears to be equipped with two rather distinct systems for beating: one involving inner-arm dyneins, the central pair and radial spokes, and the other involving outer-arm dynein alone.

Improvement of photosynthetic CO2 fixation at high light intensity through reduction of chlorophyll antenna size.

At elevated light intensities (greater than approximately 200 microE/[m2 x s]), the kinetic imbalance between the rate of photon excitation and thermally activated electron transport results in saturation of the rate of photosynthesis. Since maximum terrestrial solar radiation can reach 200 microE/(m2 x s), a significant opportunity exists to improve photosynthetic efficiency at elevated light intensities by achieving a kinetic balance between photon excitation and electron transport, especially in designed large-scale photosynthetic reactors in which a low-cost and efficient biomass production system is desired. One such strategy is a reduction in chlorophyll (chl) antenna size in relation to the reaction center that it serves. In this article, we report recent progress in this area of research. Light-saturation studies for CO2 fixation were performed on an antenna-deficient mutant of Chlamydomonas (DS521) and the wild type (DES15) with 700 ppm of CO2 in air. The light-saturated rate for CO2 assimilation in the mutant DS521 was about two times higher (187 micromol/[h x mg of chl]) than that of the wild type, DES15 (95 micromol/[h x mg of chl]). Significantly, a partial linearization of the light-saturation curve was also observed. These results confirmed that DS521 has a smaller relative chl antenna size and demonstrated that reduction of relative antenna size can improve the overall efficiency of photon utilization at higher light intensities. The antenna-deficient mutant DS521 can provide significant resistance to photoinhibition, in addition to improvement in the overall efficiency of CO2 fixation at high light. The experimental data reported herein support the idea that reduction in chl antenna size could have significant implications for both fundamental understanding of photosynthesis and potential application to improve photosynthetic CO2 fixation efficiency.

Analysis of Chlamydomonas mutants with abnormal expression of CO2 and HCO3- uptake systems.

Eukaryotic microalgae such as Chlamydomonas reinhardtii possess an inducible CO2 concentrating mechanism that operates as a very close interaction between pyrenoid-based Rubisco, various carbonic anhydrases (CAs), and inorganic carbon (Ci) transport systems. While external and internal CAs have been characterised to the molecular level, the biochemistry and molecular biology of Ci uptake mechanisms have not been elucidated. Both Ci species, CO2 and HCO3-, are taken up by the cells and chloroplasts during steady-state photosynthesis. After acclimation to limiting Ci, CO2 and HCO3- transport, measured in whole cells or chloroplasts, change their kinetic characteristics from a constitutive low-affinity state to an inducible high-affinity state. In order to learn more about the genes involved in the signal transduction pathway and in the Ci transport systems, we performed insertional mutagenesis using the arg7 gene as a selectable marker. Application of aqueous membrane inlet mass spectrometry allowed discrimination between CO2 and HCO3- uptake. Data is presented on two mutants, M46 and M21, which show severe damage to the constitutive Ci uptake systems and which are unable to induce a high-affinity state. The mutations might be either in the signal transduction pathway or in the transporters themselves. In addition, we present data that shows a very close connection between high-affinity HCO3- uptake and high-affinity NO3- uptake in cells of C. reinhardtii.

A possible role for carbonic anhydrase in the lumen of chloroplast thylakoids in green algae.

In order to understand the function of the lumen carbonic anhydrase (CA) which is bound to PSII at the lumenal side of the thylakoids in chloroplasts of eukaryotic algae, thylakoids were isolated from chloroplasts of Tetraedron minimum, Chlamydomonas noctigama, the cell wall-less mutant Chlamydomonas reinhardtii CW15, and a C. reinhardtii CW15/CIA3 mutant which lacks the lumen CA. The isolated thylakoids produced O2 on illumination and exhibited electron flow between PSII and PSI, indicating that the thylakoids were intact and the photosynthetic apparatus were functional. We could not detect any uptake of HCO3-,nor efflux of CO2, from the thylakoids upon illumination, making it improbable that the CA present in the lumen of the thylakoids would play a role in furnishing CO2 for Rubisco. We were able to determine ATP production upon illumination in isolated thylakoids. Under high inorganic carbon (Ci; 5 mM), all species showed significant amounts of ATP being produced. Under low Ci (200 M), we could not detect ATP formation from C. reinhardtii CW15/CIA3 upon illumination. This mutant was not able to survive more then 4 h of low Ci in culture. We therefore suggest that the lumen CA is not involved in the CO2 concentrating mechanism, but might play a role in the formation of a proton gradient across the thylakoid membranes.

Characterization of Chlamydomonas mutants defective in the H subunit of Mg-chelatase.

Two chlorophyll-deficient mutants of Chlamydomonas reinhardtii, chl1 and brs-1, are light sensitive and, when grown heterotrophically in the dark, accumulate protoporphyrin IX and exhibit yellow/orange pigmentation. The lesions in both mutants were mapped to the gene (CHLH) for the plastid-localized H subunit of the heterotrimeric magnesium chelatase that catalyzes the insertion of magnesium into protoporphyrin IX. The genetic defects in the mutants could be assigned to +1 frameshift mutations in exon 9 (chl1) and exon 10 (brs-1) of the CHLH gene. In both mutants, the H subunit of magnesium chelatase was undetectable, but, as shown for chl1, the steady-state levels of the I and D subunits were unaltered in comparison to wild type. The CHLH gene exhibits marked light inducibility: levels of both the mRNA and the protein product are strongly increased when cultures are shifted from from the dark into the light, suggesting that this protein may play a crucial role in the light regulation of chlorophyll biosynthesis.

Photoreceptor Current and Photoorientation in Chlamydomonas Mediated by 9-Demethylchlamyrhodopsin

Green flagellates possess rhodopsin-like photoreceptors involved in control of their behavior via generation of photocurrents across the plasma membrane. Chlamydomonas mutants blocked in retinal biosynthesis are “blind,” but they can be rescued by the addition of exogenous retinoids. Photosignaling by chlamyrhodopsin regenerated with 9-demethylretinal was investigated by recording photocurrents from single cells and cell suspensions, and by measuring phototactic orientation. The addition of a saturating concentration of this analog led to reconstitution of all receptor molecules. However, sensitivity of the photoreceptor current in cells reconstituted with the analog was smaller compared with retinal-reconstituted cells, indicating a decreased signaling efficiency of the analog receptor protein. Suppression of the photoreceptor current in double-flash experiments was smaller and its recovery faster with 9-demethylretinal than with retinal, as it would be expected from a decreased PC amplitude in the analog-reconstituted cells. Cells reconstituted with either retinal or the analog displayed negative phototaxis at low light and switched to positive one upon an increase in stimulus intensity, as opposed to the wild type. The reversal of the phototaxis direction in analog-reconstituted cells was shifted to a higher fluence rate compared with cells reconstituted with retinal, which corresponded to the decreased signaling efficiency of 9-demethylchlamyrhodopsin.

Chlamydomonas nuclear mutants that fail to assemble respiratory or photosynthetic electron transfer complexes.

We are using a molecular-genetic approach to investigate the role of nuclear genes in the biogenesis of the electron transfer complexes of mitochondria and chloroplasts. Our analysis of nuclear mutants of the green alga Chlamydomonas that are defective in respiration or photosynthesis has led to the identification of genes encoding factors required for the expression of specific organellar genes, and genes encoding structural components of the complexes.

The Chloroplast Gene ycf9 Encodes a Photosystem II (PSII) Core Subunit, PsbZ, That Participates in PSII Supramolecular Architecture

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.