Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report on the molecular and biochemical characterization of HEAT SHOCK PROTEIN 90C (HSP90C), one of the three Hsp90 chaperones encoded by the Chlamydomonas reinhardtii genome. Fractionation experiments indicate that HSP90C is a plastidic protein. In the chloroplast, HSP90C was localized to the soluble stroma fraction, but also to thylakoids and low-density membranes containing inner envelopes. HSP90C is expressed under basal conditions and is strongly induced by heat shock and moderately by light. In soluble cell extracts, HSP90C was mainly found to organize into dimers, but also into complexes of high molecular mass. Also, heterologously expressed HSP90C was mainly found in dimers, but tetramers and fewer monomers were detected, as well. HSP90C exhibits a weak ATPase activity with a Km for ATP of approximately 48 microM and a kcat of approximately 0.71 min(-1). This activity was inhibited by the Hsp90-specific inhibitor radicicol. In coimmunoprecipitation experiments, we found that HSP90C interacts with several proteins, among them plastidic HSP70B. The cellular concentration of HSP70B was found to be 2.9 times higher than that of HSP90C, giving a 4.8:1 stoichiometry of HSP70B monomers to HSP90C dimers. The strong inducibility of HSP90C by heat shock implies a role of the chaperone in stress management. Furthermore, its interaction with HSP70B suggests that, similar to their relatives in cytosol and the endoplasmic reticulum, both chaperones might constitute the core of a multichaperone complex involved in the maturation of specific client proteins, e.g. components of signal transduction pathways.
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PMID:HEAT SHOCK PROTEIN 90C is a bona fide Hsp90 that interacts with plastidic HSP70B in Chlamydomonas reinhardtii. 1599 1

The induction of a dissolved inorganic carbon (DIC) accumulating mechanism in the two algal species Scenedesmus obliquus (WT) and Chlamydomonas reinhardtii (137 c+) was physiologically characterized by monitoring DIC uptake kinetics at a low and constant DIC concentration (120-140 micromolar), after transfer from high-DIC culturing conditions. A potentiometric titration method was used to measure and calculate algal DIC uptake. Full acclimation to low-DIC conditions was obtained within a period of 90 min, after which time the DIC uptake had been increased 7 to 10 times. Experiments were also conducted in the presence of inhibitors against DIC accumulation. The inhibitor of extracellular carbonic anhydrase (CA), acetazolamide (50 micromolar), inhibited the adaptation partly, while the inhibitor of both extra- and intracellular CA, ethoxyzolamide (50 micromolar) totally inhibited the acclimation. Cycloheximide (10 micrograms per milliliter), which inhibits protein synthesis on cytoplasmic ribosomes, and vanadate (180 micromolar), which inhibits the plasmamembrane bound ATPase, also inhibited the acclimation totally. These results taken together suggest that the algae are dependent on intracellular CA, plasmamembrane bound ATPase, and de novo protein synthesis for DIC accumulation. Also, these components are more important than extracellular CA for the overall function of the DIC-accumulating mechanism.
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PMID:Induction of Inorganic Carbon Accumulation in the Unicellular Green Algae Scenedesmus obliquus and Chlamydomonas reinhardtii. 1666 60

Scenedesmus cells grown on high CO(2), when adapted to air levels of CO(2) for 4 to 6 hours in the light, formed two concentrating processes for dissolved inorganic carbon: one for utilizing CO(2) from medium of pH 5 to 8 and one for bicarbonate accumulation from medium of pH 7 to 11. Similar results were obtained with assays by photosynthetic O(2) evolution or by accumulation of dissolved inorganic carbon inside the cells. The CO(2) pump with K(0.5) for O(2) evolution of less than 5 micromolar CO(2) was similar to that previously studied with other green algae such as Chlamydomonas and was accompanied by plasmalemma carbonic anhydrase formation. The HCO(3) (-) concentrating process between pH 8 to 10 lowered the K(0.5) (DIC) from 7300 micromolar HCO(3) (-) in high CO(2) grown Scenedesmus to 10 micromolar in air-adapted cells. The HCO(3) (-) pump was inhibited by vanadate (K(i) of 150 micromolar), as if it involved an ATPase linked HCO(3) (-) transporter. The CO(2) pump was formed on low CO(2) by high-CO(2) grown cells in growth medium within 4 to 6 hours in the light. The alkaline HCO(3) (-) pump was partially activated on low CO(2) within 2 hours in the light or after 8 hours in the dark. Full activation of the HCO(3) (-) pump at pH 9 had requirements similar to the activation of the CO(2) pump. Air-grown or air-adapted cells at pH 7.2 or 9 accumulated in one minute 1 to 2 millimolar inorganic carbon in the light or 0.44 millimolar in the dark from 150 micromolar in the media, whereas CO(2)-grown cells did not accumulate inorganic carbon. A general scheme for concentrating dissolved inorganic carbon by unicellular green algae utilizes a vanadate-sensitive transporter at the chloroplast envelope for the CO(2) pump and in some algae an additional vanadate-sensitive plasmalemma HCO(3) (-) transporter for a HCO(3) (-) pump.
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PMID:Two Systems for Concentrating CO(2) and Bicarbonate during Photosynthesis by Scenedesmus. 1666 25

Intron-binding proteins in eukaryotic organelles are mainly encoded by the nuclear genome and are thought to promote the maturation of precursor RNAs. Here, we present a biochemical approach that enable the isolation of a novel nuclear-encoded protein from Chlamydomonas reinhardtii showing specific binding properties to organelle group II intron RNA. Using FPLC chromatography of chloroplast protein extracts, a 61-kDa RNA-binding protein was isolated and then tentatively identified by mass spectrometry as the chloroplast heat shock protein Cpn60. Heterologous Cpn60 protein was used in RNA protein gel mobility shift assays and revealed that the ATPase domains of Cpn60 mediates the specific binding of two group II intron RNAs, derived from the homologous chloroplast psaA gene and the heterologous mitochondrial LSU rRNA gene. The function of Cpn60 as a general organelle splicing factor is discussed.
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PMID:Chloroplast heat shock protein Cpn60 from Chlamydomonas reinhardtii exhibits a novel function as a group II intron-specific RNA-binding protein. 1687 3

T-shape radial spokes regulate flagellar beating. However, the precise function and molecular mechanism of these spokes remain unclear. Interestingly, Chlamydomonas reinhardtii flagella lacking a dimeric heat shock protein (HSP) 40 at the spokehead-spokestalk juncture appear normal in length and composition but twitch actively while cells jiggle without procession, resembling a central pair (CP) mutant. HSP40(-) cells begin swimming upon electroporation with recombinant HSP40. Surprisingly, the rescue doesn't require the signature DnaJ domain. Furthermore, the His-Pro-Asp tripeptide that is essential for stimulating HSP70 adenosine triphosphatase diverges in candidate orthologues, including human DnaJB13. Video microscopy reveals hesitance in bend initiation and propagation as well as irregular stalling and stroke switching despite fairly normal waveform. The in vivo evidence suggests that the evolutionarily conserved HSP40 specifically transforms multiple spoke proteins into stable conformation capable of mechanically coupling the CP with dynein motors. This enables 9 + 2 cilia and flagella to bend and switch to generate alternate power strokes and recovery strokes.
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PMID:Dimeric heat shock protein 40 binds radial spokes for generating coupled power strokes and recovery strokes of 9 + 2 flagella. 1822 82

Previous efforts aimed at the biochemical characterization of chloroplast HSP70B were hampered by the observation that recombinant HSP70B was inactive, i.e. incompetent of interacting with its nucleotide exchange factor CGE1. In addition, because heterologously expressed mitochondrial Hsp70 was inactive unless coexpressed with the escort protein Hep1, we wondered whether homologs of Hep1 existed in the chloroplast. Data base searches revealed that algae and higher plants indeed encode at least two HEP homologs, one predicted to be targeted to mitochondria, the others to chloroplasts. Using Chlamydomonas reinhardtii as plant model organism we demonstrate that this alga encodes an HEP homolog (termed HEP2) that is localized to the stroma. HEP2 is expressed constitutively as a low abundance protein with an apparent molecular mass of approximately 21 kDa. In cell extracts HEP2 interacts with HSP70B in an ATP-dependent fashion. Coexpression of HSP70B with HEP2 in Escherichia coli yielded high levels of CGE1-binding competent HSP70B, which also displayed ATPase activity. Inactive HSP70B was more prone to proteolysis than active HSP70B. Although inactive HSP70B interacted with HEP2, it could not be activated. Active HSP70B remained active for 48 h in the absence of HEP2, suggesting that HEP2 was not involved in maintaining HSP70B in an active state. However, some HSP70B expressed as a fusion protein with an N-terminal extension was activated when HEP2 was present during cleavage of the fusion protein, suggesting that in vivo HEP2 might be required for de novo folding of HSP70B after transit peptide cleavage.
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PMID:Assistance for a chaperone: Chlamydomonas HEP2 activates plastidic HSP70B for cochaperone binding. 1842 May 90

Outer arm dynein (OAD) of cilia and flagella contains two or three distinct heavy chains, each having a motor function. To elucidate their functional difference, we compared the in vitro motile properties of Chlamydomonas wild-type OAD containing the alpha, beta, and gamma heavy chains and three kinds of mutant OADs, each lacking one of the three heavy chains. For systematic comparison, a method was developed to introduce a biotin tag into a subunit, LC2, which served as the specific anchoring site on an avidin-coated glass surface. Wild-type OAD displayed microtubule gliding in the presence of ATP and ADP, with a maximal velocity of 5.0 mum/s, which is approximately 1/4 of the microtubule sliding velocity in the axoneme. The duty ratio was estimated to be as low as 0.08. The absence of the beta heavy chain lowered both the gliding velocity and ATPase activity, whereas the absence of the gamma heavy chain increased both activities. Strikingly, the absence of the alpha heavy chain lowered the gliding velocity but increased the ATPase activity. Thus, the three heavy chains are likely to play distinct roles and regulate each other to achieve coordinated force production.
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PMID:Systematic comparison of in vitro motile properties between Chlamydomonas wild-type and mutant outer arm dyneins each lacking one of the three heavy chains. 1912 58

Acidocalcisomes are acidic organelles with a high concentration of phosphorus present as pyrophosphate (PP(i)) and polyphosphate (poly P) complexed with calcium and other cations. The acidocalcisome membrane contains a number of pumps (Ca(2+)-ATPase, V-H(+)-ATPase, H(+)-PPase), exchangers (Na(+)/H(+), Ca(2+)/H(+)), and channels (aquaporins), while its matrix contains enzymes related to PP(i) and poly P metabolism. Acidocalcisomes have been observed in pathogenic, as well as non-pathogenic prokaryotes and eukaryotes, e.g. Chlamydomonas reinhardtii, and Dictyostelium discoideum. Some of the potential functions of the acidocalcisome are the storage of cations and phosphorus, the participation of phosphorus in PP(i) and poly P metabolism, calcium homeostasis, maintenance of intracellular pH homeostasis, and osmoregulation. In addition, acidocalcisomes resemble lysosome-related organelles (LRO) from mammalian cells in many of their properties. For example, we found that platelet dense granules, which are LROs, are very similar to acidocalcisomes. They share a similar size, acidic properties, and both contain PP(i), poly P, and calcium. Recent work that indicates that they also share the system for targeting of their membrane proteins through adaptor protein 3 reinforces this concept. The fact that acidocalcisomes interact with other organelles in parasitic protists, e.g. the contractile vacuole in Trypanosoma cruzi, and other vacuoles observed in Toxoplasma gondii, suggests that these cellular compartments may be associated with the endosomal/lysosomal pathway.
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PMID:The role of acidocalcisomes in parasitic protists. 1952 47

Assimilation of atmospheric CO2 by photosynthetic organisms such as plants, cyanobacteria and green algae, requires the production of ATP and NADPH in a ratio of 3:2. The oxygenic photosynthetic chain can function following two different modes: the linear electron flow which produces reducing power and ATP, and the cyclic electron flow which only produces ATP. Some regulation between the linear and cyclic flows is required for adjusting the stoichiometric production of high-energy bonds and reducing power. Here we explore, in the green alga Chlamydomonas reinhardtii, the onset of the cyclic electron flow during a continuous illumination under aerobic conditions. In mutants devoid of Rubisco or ATPase, where the reducing power cannot be used for carbon fixation, we observed a stimulation of the cyclic electron flow. The present data show that the cyclic electron flow can operate under aerobic conditions and support a simple competition model where the excess reducing power is recycled to match the demand for ATP.
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PMID:Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii (I) aerobic conditions. 1965 Nov 4

Large molecules require a nuclear localization signal (NLS) for translocation into the nucleus. Classical NLSs are rich in basic amino acids and they represent three groups, based on their structural features: SV40 T-antigen-type, yeast mating factor Matalpha-2-type, and bipartite NLSs. DNA-binding-with-one-finger (DOF) transcription factors play important roles in plants, and although their nuclear localization has been demonstrated in several cases, public protein localization prediction tools fail to detect NLS motifs in these proteins. Here, we demonstrate that an atypical bipartite NLS with a 17 amino acid long linker between its flanking basic regions directs Arabidopsis thaliana DOF proteins to the cell nucleus. The novel bipartite NLS is highly conserved in plant DOF transcription factors, including the single DOF protein in the green alga Chlamydomonas reinhardtii.
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PMID:A novel bipartite nuclear localization signal with an atypically long linker in DOF transcription factors. 2011 30


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