RESUMO
Certain ciliary transmembrane and membrane-associated signaling proteins export from cilia as intraflagellar transport (IFT) cargoes in a BBSome-dependent manner. Upon reaching the ciliary tip via anterograde IFT, the BBSome disassembles before being reassembled to form an intact entity for cargo phospholipase D (PLD) coupling. During this BBSome remodeling process, Chlamydomonas Rab-like 4 GTPase IFT27, by binding its partner IFT25 to form the heterodimeric IFT25/27, is indispensable for BBSome reassembly. Here, we show that IFT27 binds IFT25 in an IFT27 nucleotide-independent manner. IFT25/27 and the IFT subcomplexes IFT-A and -B are irrelevant for maintaining the stability of one another. GTP-loading onto IFT27 enhances the IFT25/27 affinity for binding to the IFT-B subcomplex core IFT-B1 entity in cytoplasm, while GDP-bound IFT27 does not prevent IFT25/27 from entering and cycling through cilia by integrating into IFT-B1. Upon at the ciliary tip, IFT25/27 cycles on and off IFT-B1 and this process is irrelevant with the nucleotide state of IFT27. During BBSome remodeling at the ciliary tip, IFT25/27 promotes BBSome reassembly independent of IFT27 nucleotide state, making postremodeled BBSomes available for PLD to interact with. Thus, IFT25/27 facilitates BBSome-dependent PLD export from cilia via controlling availability of intact BBSomes at the ciliary tip, while IFT27 nucleotide state does not participate in this regulatory event.
Assuntos
Chlamydomonas , Cílios , Nucleotídeos , Fosfolipase D , Proteínas rab de Ligação ao GTP , Cílios/química , Cílios/metabolismo , Flagelos/química , Flagelos/metabolismo , Fosfolipase D/metabolismo , Transporte Proteico , Transdução de Sinais , Chlamydomonas/citologia , Chlamydomonas/enzimologia , Chlamydomonas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Difosfato/metabolismoRESUMO
Photosynthetic organisms have developed a regulation mechanism called state transition (ST) to rapidly adjust the excitation balance between the two photosystems by light-harvesting complex II (LHCII) movement. Though many researchers have assumed coupling of the dynamic transformations of the thylakoid membrane with ST, evidence of that remains elusive. To clarify the above-mentioned coupling in a model organism Chlamydomonas, here we used two advanced microscope techniques, the excitation-spectral microscope (ESM) developed recently by us and the superresolution imaging based on structured-illumination microscopy (SIM). The ESM observation revealed ST-dependent spectral changes upon repeated ST inductions. Surprisingly, it clarified a less significant ST occurrence in the region surrounding the pyrenoid, which is a subcellular compartment specialized for the carbon-fixation reaction, than that in the other domains. Further, we found a species dependence of this phenomenon: 137c strain showed the significant intracellular inhomogeneity of ST occurrence, whereas 4A+ strain hardly did. On the other hand, the SIM observation resolved partially irreversible fine thylakoid transformations caused by the ST-inducing illumination. This fine, irreversible thylakoid transformation was also observed in the STT7 kinase-lacking mutant. This result revealed that the fine thylakoid transformation is not induced solely by the LHCII phosphorylation, suggesting the highly susceptible nature of the thylakoid ultrastructure to the photosynthetic light reactions.
Assuntos
Chlamydomonas , Complexos de Proteínas Captadores de Luz , Complexo de Proteína do Fotossistema II , Tilacoides , Chlamydomonas/enzimologia , Chlamydomonas/efeitos da radiação , Luz , Complexos de Proteínas Captadores de Luz/química , Fosforilação , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/química , Tilacoides/enzimologia , Tilacoides/efeitos da radiaçãoRESUMO
Microalgae are photosynthetic unicellular organisms that can be found in very different environments, both terrestrial and marine, including extreme environments such as cold, hot and high/low salinity [...].
Assuntos
Microalgas/enzimologia , Microalgas/genética , Microalgas/metabolismo , Biomassa , Chlamydomonas/enzimologia , Chlamydomonas/metabolismo , FotossínteseRESUMO
Light/dark cycling is an inherent condition of outdoor microalgae cultivation, but is often unfavorable for lipid accumulation. This study aims to identify promising targets for metabolic engineering of improved lipid accumulation under outdoor conditions. Consequently, the lipid-rich mutant Chlamydomonas sp. KOR1 was developed through light/dark-conditioned screening. During dark periods with depressed CO2 fixation, KOR1 shows rapid carbohydrate degradation together with increased lipid and carotenoid contents. KOR1 was subsequently characterized with extensive mutation of the ISA1 gene encoding a starch debranching enzyme (DBE). Dynamic time-course profiling and metabolomics reveal dramatic changes in KOR1 metabolism throughout light/dark cycles. During light periods, increased flux from CO2 through glycolytic intermediates is directly observed to accompany enhanced formation of small starch-like particles, which are then efficiently repartitioned in the next dark cycle. This study demonstrates that disruption of DBE can improve biofuel production under light/dark conditions, through accelerated carbohydrate repartitioning into lipid and carotenoid.
Assuntos
Proteínas de Algas/metabolismo , Metabolismo dos Carboidratos , Carotenoides/metabolismo , Chlamydomonas/metabolismo , Metabolismo dos Lipídeos , Amido/metabolismo , Chlamydomonas/enzimologia , Microalgas/enzimologia , Microalgas/metabolismoRESUMO
We report a structure-based biological approach to identify the proton-transfer pathway in photosystem II. First, molecular dynamics (MD) simulations were conducted to analyze the H-bond network that may serve as a Grotthuss-like proton conduit. MD simulations show that D1-Asp61, the H-bond acceptor of H2O at the Mn4CaO5 cluster (W1), forms an H-bond via one water molecule with D1-Glu65 but not with D2-Glu312. Then, D1-Asp61, D1-Glu65, D2-Glu312, and the adjacent residues, D1-Arg334, D2-Glu302, and D2-Glu323, were thoroughly mutated to the other 19 residues, i.e., 114 Chlamydomonas chloroplast mutant cells were generated. Mutation of D1-Asp61 was most crucial. Only the D61E and D61C cells grew photoautotrophically and exhibit O2-evolving activity. Mutations of D2-Glu312 were less crucial to photosynthetic growth than mutations of D1-Glu65. Quantum mechanical/molecular mechanical calculations indicated that in the PSII crystal structure, the proton is predominantly localized at D1-Glu65 along the H-bond with D2-Glu312, i.e., pKa(D1-Glu65) > pKa(D2-Glu312). The potential-energy profile shows that the release of the proton from D1-Glu65 leads to the formation of the two short H-bonds between D1-Asp61 and D1-Glu65, which facilitates downhill proton transfer along the Grotthuss-like proton conduit in the S2 to S3 transition. It seems possible that D1-Glu65 is involved in the dominant pathway that proceeds from W1 via D1-Asp61 toward the thylakoid lumen, whereas D2-Glu312 and D1-Arg334 may be involved in alternative pathways in some mutants.
Assuntos
Chlamydomonas/enzimologia , Modelos Químicos , Modelos Moleculares , Oxigênio/química , Complexo de Proteína do Fotossistema II/química , Prótons , Chlamydomonas/genética , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismoAssuntos
Chlamydomonas/enzimologia , Chlamydomonas/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Família Multigênica , Catálise , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/genética , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , DNA de Cinetoplasto/genética , DNA de Cinetoplasto/metabolismo , Células Eucarióticas/enzimologia , Evolução Molecular , Interações Hospedeiro-Patógeno/genética , Família Multigênica/fisiologia , Especificidade por SubstratoRESUMO
In land plants and algae, the Calvin-Benson (CB) cycle takes place in the chloroplast, a specialized organelle in which photosynthesis occurs. Thioredoxins (TRXs) are small ubiquitous proteins, known to harmonize the two stages of photosynthesis through a thiol-based mechanism. Among the 11 enzymes of the CB cycle, the TRX target phosphoribulokinase (PRK) has yet to be characterized at the atomic scale. To accomplish this goal, we determined the crystal structures of PRK from two model species: the green alga Chlamydomonas reinhardtii (CrPRK) and the land plant Arabidopsis thaliana (AtPRK). PRK is an elongated homodimer characterized by a large central ß-sheet of 18 strands, extending between two catalytic sites positioned at its edges. The electrostatic surface potential of the catalytic cavity has both a positive region suitable for binding the phosphate groups of substrates and an exposed negative region to attract positively charged TRX-f. In the catalytic cavity, the regulatory cysteines are 13 Å apart and connected by a flexible region exclusive to photosynthetic eukaryotes-the clamp loop-which is believed to be essential for oxidation-induced structural rearrangements. Structural comparisons with prokaryotic and evolutionarily older PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and an increased number of random-coiled regions, suggesting that a general loss in structural rigidity correlates with gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes.
Assuntos
Arabidopsis , Chlamydomonas , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fotossíntese/fisiologia , Proteínas de Plantas/química , Arabidopsis/química , Arabidopsis/enzimologia , Chlamydomonas/química , Chlamydomonas/enzimologia , Cristalografia , Modelos Moleculares , Oxirredução , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/químicaRESUMO
Many secreted peptides used for cell-cell communication require conversion of a C-terminal glycine to an amide for bioactivity. This reaction is catalyzed only by the integral membrane protein peptidylglycine α-amidating monooxygenase (PAM). PAM has been highly conserved and is found throughout the metazoa; PAM-like sequences are also present in choanoflagellates, filastereans, unicellular and colonial chlorophyte green algae, dinoflagellates and haptophytes. Recent studies have revealed that in addition to playing a key role in peptidergic signaling, PAM also regulates ciliogenesis in vertebrates, planaria and chlorophyte algae, and is required for the stability of actin-based microvilli. Here we briefly introduce the basic principles involved in ciliogenesis, the sequential reactions catalyzed by PAM and the trafficking of PAM through the secretory and endocytic pathways. We then discuss the multi-faceted roles this enzyme plays in the formation and maintenance of cytoskeleton-based cellular protrusions and propose models for how PAM protein and amidating activity might contribute to ciliogenesis. Finally, we consider why some ciliated organisms lack PAM, and discuss the potential ramifications of ciliary localized PAM for the endocrine features commonly observed in patients with ciliopathies.
Assuntos
Chlamydomonas/enzimologia , Cílios/metabolismo , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/metabolismo , Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Actinas/metabolismo , Chlamydomonas/citologia , Chlamydomonas/metabolismo , Chlamydomonas/ultraestrutura , Cílios/ultraestrutura , Oxigenases de Função Mista/análise , Modelos Moleculares , Complexos Multienzimáticos/análise , Proteínas de Plantas/análise , Biossíntese de Proteínas , Transporte Proteico , Transdução de SinaisRESUMO
Members of the MAPK superfamily are known as key regulators of ciliogenesis. Long flagellar (LF) 4, a MAPK-related kinase in Chlamydomonas, is the first kinase that was implicated in ciliary assembly and length. However, little is known about its cellular properties, regulation, and molecular functions. LF4 is localized both in the flagella and cell body with enrichment at the 2 basal bodies, shown by super-resolution microscopy. LF4 is constitutively phosphorylated at T159 at the kinase activation loop and remains at the basal bodies during flagellar assembly. Gene mutations that affect the kinase activity or T159 phosphorylation alter the localization of LF4 at the basal bodies, and the mutants fail to rescue lf4-3, a null mutant. LF4 does not affect the velocities of intraflagellar transport (IFT). However, LF4 null mutation induces accumulation of IFT proteins in the flagellum and reduces the phosphorylation of the kinesin-II subunit FLA8/KIF3B, indicating that LF4 negatively regulates IFT entry. Furthermore, LF2, a cell cycle-related kinase, and LF3, a novel protein, are required for LF4 phosphorylation. Our study demonstrates that LF4 is likely a constitutively active kinase that is regulated by LF2 and regulates IFT entry at the basal bodies to control flagellar assembly and length.-Wang, Y., Ren, Y., Pan, J. Regulation of flagellar assembly and length in Chlamydomonas by LF4, a MAPK-related kinase.
Assuntos
Chlamydomonas/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flagelos/enzimologia , Proteínas de Plantas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chlamydomonas/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Flagelos/genética , Fosforilação , Proteínas de Plantas/genéticaRESUMO
Carbonic anhydrases (CAs) are a class of zinc-containing metalloenzymes that can reversibly catalyse the hydration reaction of carbon dioxide. Antarctic algae are the most critical component of the Antarctic ecosystem; algae can enter the carbon cycle food chain by fixing carbon dioxide from the air. In this study, the complete open reading frames (ORFs) of CA1 (GenBank ID KY826431), CA2 (GenBank ID KY826432), and CA3 (GenBank ID KY826433), encoding CAs in the Antarctic ice microalga Chlamydomonas. sp. ICE-L, were successfully cloned using reverse transcription-polymerase chain reaction (RT-PCR). In addition, the expression patterns of CAs under blue light, under UV light, and in the dark were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The CA1, CA2, and CA3 ORFs encode proteins of 376, 430, and 419 amino acids, respectively. Phylogenetic analysis revealed that all amino acid sequences showed high homology with those of C. sp. ICE-L. There are six types of algal CAs; we hypothesised that the CAs studied here are most likely α-CAs. Expression analysis showed that the transcription level of the CAs was influenced by both UV light and blue light. These findings provide additional insight into the molecular mechanisms of CAs and will accelerate the development of CAs for applications in agriculture and environmental governance.
Assuntos
Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Chlamydomonas/enzimologia , Clonagem Molecular/efeitos dos fármacos , Proteínas de Algas/química , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Anidrases Carbônicas/química , Chlamydomonas/genética , Evolução Molecular , Modelos Moleculares , Fases de Leitura Aberta , Filogenia , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
The objective of this work was to characterize photosynthetic ferredoxin from the Antarctic green alga Chlamydomonas sp. UWO241, a key enzyme involved in distributing photosynthetic reducing power. We hypothesize that ferredoxin possesses characteristics typical of cold-adapted enzymes, namely increased structural flexibility and high activity at low temperatures, accompanied by low stability at moderate temperatures. To address this objective, we purified ferredoxin from UWO241 and characterized the temperature dependence of its enzymatic activity and protein conformation. The UWO241 ferredoxin protein, RNA, and DNA sequences were compared with homologous sequences from related organisms. We provide evidence for the duplication of the main ferredoxin gene in the UWO241 nuclear genome and the presence of two highly similar proteins. Ferredoxin from UWO241 has both high activity at low temperatures and high stability at moderate temperatures, representing a novel class of cold-adapted enzymes. Our study reveals novel insights into how photosynthesis functions in the cold. The presence of two distinct ferredoxin proteins in UWO241 could provide an adaptive advantage for survival at cold temperatures. The primary amino acid sequence of ferredoxin is highly conserved among photosynthetic species, and we suggest that subtle differences in sequence can lead to significant changes in activity at low temperatures.
Assuntos
Adaptação Fisiológica , Chlamydomonas/fisiologia , Temperatura Baixa , Fotossíntese , Sequência de Aminoácidos , Regiões Antárticas , Chlamydomonas/enzimologia , Chlamydomonas/genética , Ferredoxinas/química , Ferredoxinas/metabolismo , Genoma , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transcriptoma/genéticaRESUMO
The psychrophilic microalga, Chlamydomonas sp. ICE-L, isolated from floating ice in the Antarctic, one of the most highly UV exposed ecosystems on Earth, displays an efficient DNA photorepair capacity. Here, the first known (6-4) photolyase gene (6-4CiPhr) from C. sp. ICE-L was identified. The 6-4CiPhr encoded 559-amino acid polypeptide with a pI of 8.86, and had a predicted Mw of 64.2â¯kDa. Real-time PCR was carried out to investigate the response of 6-4CiPhr to UVB exposure. The transcription of 6-4CiPhr was up-regulated continuously within 6â¯h, achieving a maximum of 62.7-fold at 6â¯h. Expressing 6-4CiPhr in a photolyase-deficient Escherichia coli strain improved survival rate of the strain. In vitro activity assays of purified protein demonstrated that 6-4CiPhr was a photolyase with 6-4PP repair activity. These findings improve understanding of photoreactivation mechanisms of (6-4) photolyase.
Assuntos
Chlamydomonas , Desoxirribodipirimidina Fotoliase , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Proteínas de Plantas , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta , Regulação para Cima/efeitos da radiação , Chlamydomonas/enzimologia , Chlamydomonas/genética , Desoxirribodipirimidina Fotoliase/biossíntese , Desoxirribodipirimidina Fotoliase/química , Desoxirribodipirimidina Fotoliase/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The assembly of membranous extensions such as microvilli and cilia in polarized cells is a tightly regulated, yet poorly understood, process. Peptidylglycine α-amidating monooxygenase (PAM), a membrane enzyme essential for the synthesis of amidated bioactive peptides, was recently identified in motile and non-motile (primary) cilia and has an essential role in ciliogenesis in Chlamydomonas, Schmidtea and mouse. In mammalian cells, changes in PAM levels alter secretion and organization of the actin cytoskeleton. Here we show that lack of Pam in zebrafish recapitulates the lethal edematous phenotype observed in Pam -/- mice and reveals additional defects. The pam -/- zebrafish embryos display an initial striking loss of microvilli and subsequently impaired ciliogenesis in the pronephros. In multiciliated mouse tracheal epithelial cells, vesicular PAM staining colocalizes with apical actin, below the microvilli. In PAM-deficient Chlamydomonas, the actin cytoskeleton is dramatically reorganized, and expression of an actin paralogue is upregulated. Biochemical assays reveal that the cytosolic PAM C-terminal domain interacts directly with filamentous actin but does not alter the rate of actin polymerization or disassembly. Our results point to a critical role for PAM in organizing the actin cytoskeleton during development, which could in turn impact both microvillus formation and ciliogenesis.
Assuntos
Actinas/metabolismo , Linhagem Celular/metabolismo , Chlamydomonas/enzimologia , Cílios/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Proteínas de Algas/química , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Animais , Sítios de Ligação , Técnicas de Silenciamento de Genes , Camundongos , Microvilosidades , Oxigenases de Função Mista/química , Complexos Multienzimáticos/química , Domínios Proteicos , Traqueia/citologia , Traqueia/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The α-carbonic anhydrase (α-CA) is a zinc ion-containing enzyme that catalyzes the hydration of carbon dioxide. In this paper, a full-length α-CA gene was cloned from Chlamydomonas sp. ICE-L using RT-PCR and RACE-PCR for bioinformatic analysis. The α-CA open reading frame obtained by PCR was cloned into a vector and transformed into Escherichia coli to generate α-CA-producing bacteria. The α-CA was highly expressed upon induction with isopropyl-ß-d-thiogalactoside (IPTG) at a final concentration of 0.8 mM. A single band with a molecular weight of approximate 40 kDa expressed in the recombinant E. coli strain harboring the α-CA vector was observed in SDS-PAGE analysis. The carbon dioxide hydration activity and esterase activity of α-CA expressed by the recombinant strain were 0.404 U/mg and 0.319 U, respectively. In addition, three conditions, temperature, salinity and UVB radiation exposure, were selected to analyze α-CA transcription levels by qRT-PCR. The results suggested UVB exposure increased the expression of relative mRNA; meanwhile, the α-CA mRNA expression was rapidly induced by temperature and salinity stress, indicating that Chlamydomonas sp. ICE-L might modulate the α-CA mRNA expression to adapt to the extreme environments.
Assuntos
Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Chlamydomonas/enzimologia , Dióxido de Carbono/metabolismo , Chlamydomonas/genética , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Filogenia , TemperaturaRESUMO
The metabolism of glycerol-3-phosphate (G3P) is important for environmental stress responses by eukaryotic microalgae. G3P is an essential precursor for glycerolipid synthesis and the accumulation of triacylglycerol (TAG) in response to nutrient starvation. G3P dehydrogenase (GPDH) mediates G3P synthesis, but the roles of specific GPDH isoforms are currently poorly understood. Of the five GPDH enzymes in the model alga Chlamydomonas reinhardtii, GPD2 and GPD3 were shown to be induced by nutrient starvation and/or salt stress. Heterologous expression of GPD2, a putative chloroplastic GPDH, and GPD3, a putative cytosolic GPDH, in a yeast gpd1Δ mutant demonstrated the functionality of both enzymes. C. reinhardtii knockdown mutants for GPD2 and GPD3 showed no difference in growth but displayed significant reduction in TAG concentration compared with the wild type in response to phosphorus or nitrogen starvation. Overexpression of GPD2 and GPD3 in C. reinhardtii gave distinct phenotypes. GPD2 overexpression lines showed only subtle metabolic phenotypes and no significant alteration in growth. In contrast, GPD3 overexpression lines displayed significantly inhibited growth and chlorophyll concentration, reduced glycerol concentration, and changes to lipid composition compared with the wild type, including increased abundance of phosphatidic acids but reduced abundance of diglycerides, triglycerides, and phosphatidylglycerol lipids. This may indicate a block in the downstream glycerolipid metabolism pathway in GPD3 overexpression lines. Thus, lipid engineering by GPDH modification may depend on the activities of other downstream enzyme steps. These results also suggest that GPD2 and GPD3 GPDH isoforms are important for nutrient starvation-induced TAG accumulation but have distinct metabolic functions.
Assuntos
Chlamydomonas/enzimologia , Glicerolfosfato Desidrogenase/metabolismo , Metabolismo dos Lipídeos , Biomassa , Carboidratos/análise , Chlamydomonas/genética , Chlamydomonas/crescimento & desenvolvimento , Clorofila/metabolismo , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Genes de Plantas , Teste de Complementação Genética , Glicerol/metabolismo , Mutação/genética , Fenótipo , Plantas Geneticamente Modificadas , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Amido/metabolismo , Estresse Fisiológico/genéticaRESUMO
The pathways controlling cilium biogenesis in different cell types have not been fully elucidated. We recently identified peptidylglycine α-amidating monooxygenase (PAM), an enzyme required for generating amidated bioactive signaling peptides, in Chlamydomonas and mammalian cilia. Here, we show that PAM is required for the normal assembly of motile and primary cilia in Chlamydomonas, planaria and mice. Chlamydomonas PAM knockdown lines failed to assemble cilia beyond the transition zone, had abnormal Golgi architecture and altered levels of cilia assembly components. Decreased PAM gene expression reduced motile ciliary density on the ventral surface of planaria and resulted in the appearance of cytosolic axonemes lacking a ciliary membrane. The architecture of primary cilia on neuroepithelial cells in Pam-/- mouse embryos was also aberrant. Our data suggest that PAM activity and alterations in post-Golgi trafficking contribute to the observed ciliogenesis defects and provide an unanticipated, highly conserved link between PAM, amidation and ciliary assembly.
Assuntos
Chlamydomonas/enzimologia , Cílios/metabolismo , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/metabolismo , Biogênese de Organelas , Animais , Técnicas de Silenciamento de Genes , Camundongos/embriologia , Camundongos Knockout , Oxigenases de Função Mista/genética , Complexos Multienzimáticos/genética , Planárias/enzimologiaRESUMO
Calmodulin (CaM) is a Ca2+-binding protein that plays a role in several Ca2+ signaling pathways, which dynamically regulates the activities of hundreds of proteins. The ice alga Chlamydomonas sp. ICE-L, which has the ability to adapt to extreme polar conditions, is a crucial primary producer in Antarctic ecosystem. This study hypothesized that Cam helps the ICE-L to adapt to the fluctuating conditions in the polar environment. It first verified the overall length of Cam, through RT-PCR and RACE-PCR, based on partial Cam transcriptome library of ICE-L. Then, the nucleotide and predicted amino acid sequences were, respectively, analyzed by various bioinformatics approaches to gain more insights into the computed physicochemical properties of the CaM. Potential involvements of Cam in responding to certain stimuli (i.e., UVB radiation, high salinity, and temperature) were investigated by differential expression, measuring its transcription levels by means of quantitative RT-PCR. Results showed that CaM was indeed inducible and regulated by high UVB radiation, high salinity, and nonoptimal temperature conditions. Different conditions had different expression tendencies, which provided an important basis for investigating the adaptation mechanism of Cam in ICE-L.
Assuntos
Calmodulina/análise , Calmodulina/genética , Chlamydomonas/enzimologia , Perfilação da Expressão Gênica , Regiões Antárticas , Calmodulina/química , Chlamydomonas/efeitos dos fármacos , Chlamydomonas/genética , Chlamydomonas/efeitos da radiação , Clonagem Molecular , Biologia Computacional , Pressão Osmótica , Reação em Cadeia da Polimerase , Salinidade , Temperatura , Raios UltravioletaRESUMO
Peroxisomes are thought to have played a key role in the evolution of metabolic networks of photosynthetic organisms by connecting oxidative and biosynthetic routes operating in different compartments. While the various oxidative pathways operating in the peroxisomes of higher plants are fairly well characterized, the reactions present in the primitive peroxisomes (microbodies) of algae are poorly understood. Screening of a Chlamydomonas insertional mutant library identified a strain strongly impaired in oil remobilization and defective in Cre05.g232002 (CrACX2), a gene encoding a member of the acyl-CoA oxidase/dehydrogenase superfamily. The purified recombinant CrACX2 expressed in Escherichia coli catalyzed the oxidation of fatty acyl-CoAs into trans-2-enoyl-CoA and produced H2 O2 . This result demonstrated that CrACX2 is a genuine acyl-CoA oxidase, which is responsible for the first step of the peroxisomal fatty acid (FA) ß-oxidation spiral. A fluorescent protein-tagging study pointed to a peroxisomal location of CrACX2. The importance of peroxisomal FA ß-oxidation in algal physiology was shown by the impact of the mutation on FA turnover during day/night cycles. Moreover, under nitrogen depletion the mutant accumulated 20% more oil than the wild type, illustrating the potential of ß-oxidation mutants for algal biotechnology. This study provides experimental evidence that a plant-type FA ß-oxidation involving H2 O2 -producing acyl-CoA oxidation activity has already evolved in the microbodies of the unicellular green alga Chlamydomonas reinhardtii.
Assuntos
Acil-CoA Oxidase/metabolismo , Chlamydomonas/enzimologia , Chlamydomonas/metabolismo , Peroxissomos/metabolismo , Chlamydomonas/genética , Peróxido de Hidrogênio/metabolismo , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Nitrogênio/metabolismo , OxirreduçãoRESUMO
Introducing components of algal carbon concentrating mechanisms (CCMs) into higher plant chloroplasts could increase photosynthetic productivity. A key component is the Rubisco-containing pyrenoid that is needed to minimise CO2 retro-diffusion for CCM operating efficiency. Rubisco in Arabidopsis was re-engineered to incorporate sequence elements that are thought to be essential for recruitment of Rubisco to the pyrenoid, namely the algal Rubisco small subunit (SSU, encoded by rbcS) or only the surface-exposed algal SSU α-helices. Leaves of Arabidopsis rbcs mutants expressing 'pyrenoid-competent' chimeric Arabidopsis SSUs containing the SSU α-helices from Chlamydomonas reinhardtii can form hybrid Rubisco complexes with catalytic properties similar to those of native Rubisco, suggesting that the α-helices are catalytically neutral. The growth and photosynthetic performance of complemented Arabidopsis rbcs mutants producing near wild-type levels of the hybrid Rubisco were similar to those of wild-type controls. Arabidopsis rbcs mutants expressing a Chlamydomonas SSU differed from wild-type plants with respect to Rubisco catalysis, photosynthesis and growth. This confirms a role for the SSU in influencing Rubisco catalytic properties.
Assuntos
Arabidopsis/genética , Chlamydomonas/enzimologia , Teste de Complementação Genética , Mutação/genética , Subunidades Proteicas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Biocatálise , Clorofila/metabolismo , Fluorescência , Regulação da Expressão Gênica de Plantas , Isoenzimas/metabolismo , Fenótipo , Fotossíntese , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Subunidades Proteicas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribulose-Bifosfato Carboxilase/químicaRESUMO
Arctic Chlamydomonas sp. is a dominant microalgal strain in cold or frozen freshwater in the Arctic region. The full-length open reading frame of the omega-6 fatty acid desaturase gene (AChFAD6) was obtained from the transcriptomic database of Arctic Chlamydomonas sp. from the KOPRI culture collection of polar micro-organisms. Amino acid sequence analysis indicated the presence of three conserved histidine-rich segments as unique characteristics of omega-6 fatty acid desaturases, and three transmembrane regions transported to plastidic membranes by chloroplast transit peptides in the N-terminal region. The AChFAD6 desaturase activity was examined by expressing wild-type and V254A mutant (Mut-AChFAD6) heterologous recombinant proteins. Quantitative gas chromatography indicated that the concentration of linoleic acids in AChFAD6-transformed cells increased more than 3-fold [6.73 ± 0.13 mg g-1 dry cell weight (DCW)] compared with cells transformed with vector alone. In contrast, transformation with Mut-AChFAD6 increased the concentration of oleic acid to 9.23 ± 0.18 mg g-1 DCW, indicating a change in enzymatic activity to mimic that of stearoyl-CoA desaturase. These results demonstrate that AChFAD6 of Arctic Chlamydomonas sp. increases membrane fluidity by enhancing denaturation of C18 fatty acids and facilitates production of large quantities of linoleic fatty acids in prokaryotic expression systems.