Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Halotolerant Green Alga Dunaliella tertiolecta

A Ca²⁺-dependent protein kinase (CDPK) that has been partiallypurified and characterized previously [Yuasa and Muto (1992)Arch. Biochem. Biophys. 296: 175] was further purified to about20,000-fold from the soluble fraction of Dunaliella tertiolecta.The enzyme preparation contained 60- and 52-kDa polypeptidesboth of which phosphorylated casein as a substrate. Both polypeptidesshowed a Ca²⁺-dependent increase in mobility during SDS-PAGEand ⁴⁵Ca²⁺-binding activity after SDS-PAGE and electroblottingonto a nitrocellulose membrane, suggesting that both the 60-and 52-kDa CDPKs directly bind Ca²⁺. The protein kinase inhibitors,K-252a and staurosporine, inhibited the CDPK competitively withrespect to ATP. An antibody raised against the 60-kDa CDPK crossreactedwith both the 60- and 52-kDa polypeptides. Both molecular specieswere autophosphorylated in the presence of Ca²⁺, and a highlyphosphorylated 80-kDa band appeared in addition to these phosphorylatedbands at 60 and 52 kDa in SDS-PAGE. However, the specific activityof CDPK was not changed by prior autophosphorylation when theautophosphorylated enzyme was assayed as a mixture of thesephosphorylated molecular species. Only the 60-kDa polypeptidewas immunodetected in subcellular fractions of Dunaliella cells.The 52-kDa polypeptide increased during storage of the enzyme.These results suggest that the 52-kDa polypeptide is a proteolyticartifact produced during purification. Immunoreactive bandsof 60-kDa were detected in extracts of several green algae butnot in extracts of higher plants or a brown alga. ¹This research was partly supported by Grants-in-Aid from theMinistry of Education, Science and Culture, Japan (No. 06454013and 06304023) and Research Fellowship of the Japan Society forthe Promotion of Science for Young Sciencists. ²Research Fellow (PD) of the Japan Society for the Promotionof Science.     

Salt-dependent Protein Composition in the Halotolerant Green Alga,Dunaliella parva

The extremely halotolerant green alga, Dunaliella parva, tolerates salt concentrations from 0.3 to 3.0 M NaCl. Effects of long-term adaptation to five distinct salinities were analyzed. Salt-dependent differences of physiological parameters such as growth rate, pigments, quantitative protein contents, and gas exchange were measured; furthermore the qualitative protein composition in salt-adapted cells was investigated using SDS-polyacrylamide gel electrophoresis. Proteins of apparent molecular masses of 26, 35, 39, 50, and 63 kDa were induced or intensified with an increase in external sodium chloride concentration whereas proteins of 85 and 101 kDa were diminished in high salt algae. After selective staining, four modifications of glycoproteins were observed. A glycoprotein of 96 kDa was produced exclusively in low salt cells whereas glycosylations of 105, 135, and 260 kDa were induced by high salt concentrations.     

A 150 Kilodalton Cell Surface Protein Is Induced by Salt in the Halotolerant Green Alga Dunaliella salina

Dunaliella salina is an extremely halotolerant, unicellular, green alga lacking a rigid cell wall. Osmotic adaptation to high salinities is based on the accumulation of glycerol. To uncover other functions responsible for halotolerance, protein profiles of algae continuously grown in different salinities were compared. A 150 kilodalton protein (p 150) increased in amount with salt concentration. Furthermore, when the cells were subjected to drastic hyperosmotic shocks, p150 started to rise long after completion of the osmotic response but coincident with reinitiation of cell proliferation. Cells with an initially higher level of p150 resumed growth faster than cells with a lower level of the protein. Addition of cycloheximide early after hyperosmotic shock prevented the rise in p150, indicating this rise was due to de novo synthesis of the protein. These observations suggest that p150 is a saltinduced protein required for proliferation of the cells in saline media. p150 was purified to homogeneity and found to be a detergent-soluble glycoprotein. Polyclonal antibodies against p150 recognized a single protein component in D. salina crude extracts. A high M_r cross-reacting protein was also observed in another Dunaliella strain, D. bardawil. Immunoelectron microscopy localized p150 to the cell surface.     

Ion Content of the Halotolerant Alga Dunaliella salina

The intracellular concentration of the major ions in Dunaliellasalina cells were determined, following the removal of extracellularions by ion-exchange minicolumns. Log phase cells, grown inmedia containing 1–4 molar NaCl, contained 30–50mM chloride and 200–350 mM magnesium (5 mM in medium).Phosphorus, which is present intracellularly mostly as polyphosphate,was present in amounts of 60–100 fmoles per cell, equivalentto a concentration of 600–1,000 mM (0.2 mM in medium).Previous data indicated that such cells contained 20–40mM Na⁺, 150–300mM K⁺, 20mM SO^2–₄, and very low concentrationsof Ca2⁺ and charged nitrogenous compounds. Mg²⁺ and K⁺ seemto serve as the major counter ions for the intracellular negativecharge present in the massively accumulated polyphosphates.The former accounts for about 2/3 of the required positive charge.This is supported by the observation that limitation in thephosphate or K⁺ supply in the medium lead to a parallel decreasein the accumulation of intracellular phosphorus, Mg²⁺ or K⁺. ¹Present address: Department of Vegetables, The Volcani Center,Bet-Dagan 50250, Israel. (Received June 13, 1988; Accepted August 25, 1988)     

Genome Size Affects Fitness in the Eukaryotic Alga Dunaliella tertiolecta

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Polyphosphate Hydrolysis within Acidic Vacuoles in Response to Amine-Induced Alkaline Stress in the Halotolerant Alga Dunaliella salina

The location and mobilization of polyphosphates in response to an amine-induced alkaline stress were studied in the halotolerant alga Dunaliella salina. The following observations suggest that polyphosphates accumulate in acidic vacuoles: (a) Accumulation of large amounts of polyphosphates is manifested as intravacuolar dense osmiophilic bodies in electron micrographs. (b) Uptake of amines into the vacuoles induces massive hydrolysis of polyphosphates, demonstrated by in vivo ³¹P-nuclear magnetic resonance, and by analysis of hydrolytic products on thin layer chromatograms. The analysis indicates that: (a) Polyphosphate hydrolysis is kinetically correlated with amine accumulation and with the recovery of cytoplasmic pH. (b) The major hydrolytic product is tripolyphosphate. (c) The peak position of the tripolyphosphate terminal phosphate in nuclear magnetic resonance spectra is progressively shifted as the cells recover, indicating that the pH inside the vacuoles increases while the pH in the cytoplasm decreases. (d) In lysed cell preparations, in which vacuoles become exposed to the external pH, mild alkalinization in the absence of amines induces polyphosphate hydrolysis to tripolyphosphates. It is suggested that amine accumulation within vacuoles activates a specific phosphatase, which hydrolyzes long-chain polyphosphates to tripolyphosphates. The hydrolysis increases the capacity of the vacuoles to sequester amines from the cytoplasm probably by releasing protons required to buffer the amine, and leads to recovery of cytoplasmic pH. Thus, polyphosphate hydrolysis provides a high-capacity buffering system that sustains amine compartmentation into vacuoles and protects cytoplasmic pH.     

Fatty Acid Acylated Proteins of the Halotolerant Alga Dunaliella salina

The unicellular, wall-less alga Dunaliella salina has been shown to contain an array of proteins modified by the covalent attachment of fatty acids. Myristic acid (14:0) comprised approximately 80% by weight of the protein-linked acyl groups in samples derived from cells cultured in medium containing 1.7 molar NaCl and 93% in samples from cells grown in medium containing 3.0 molar NaCl. Palmitic and stearic acids accounted for most of the remaining protein-bound acyl chains. Approximately 0.2% of the incorporated radioactivity was estimated to be in linkage with protein. The bulk of acyl chains (about 99%) were resistant to cleavage by alkali, indicating a preponderance of amide bonding. The sodium dodecyl sulfate-polyacrylamide electrophoresis labeling pattern of proteins from [³H]myristic-labeled cells was significantly different from that of proteins from cells exposed to [³H]palmitate. The appearance of radioactivity in certain proteins was also influenced by the salinity of the culture medium. Thus growth in moderate (1.7 molar) salt favored the acylation of a 48-kilodalton polypeptide whereas in high (3.0 molar) salt, a 17-kilodalton polypeptide was more heavily labeled.     

Dynamics of Photosystem II and Its Light Harvesting System in Response to Light Changes in the Halotolerant Alga Dunaliella salina

A photosystem two (PSII) core complex consisting of five major polypeptides (47, 40, 32, 30, and 10 kilodaltons) and a light harvesting chlorophyll a/b complex (LHC-2) have been isolated from the halotolerant alga Dunaliella salina. The chlorophyll and polypeptide composition of both complexes were compared in illuminated and dark-adapted cultures. Dark adaptation is accompanied by a decrease in the chlorophyll a to chlorophyll b (Chl a/Chl b) ratio of intact thylakoids without any change in total chlorophyll. These changes occur with a half-time of 3 hours and are reversed upon reillumination. Analyses of PSII enriched membrane fragments suggest that the decrease in the Chl a/Chl b is due partly to an increase in the Chl b content of LHC-2 and partly to changes in the relative levels of the two complexes. Apparently during dark adaptation there is: (a) a net synthesis of chlorophyll b, (b) removal of PSII core complexes resulting in a 2-fold drop in the PSII cores to LHC-2 chlorophyll ratio. These changes should dramatically increase the light harvesting capacity of the remaining PSII reaction centers. Presumably this adjustment of antenna size and composition is a physiological mechanism necessary for responding to shade conditions. Also detected, using ³²P, are light-induced phosphorylation of the LHC-2 (consistent with the ability to undergo State transitions) and of the 40 and 30 kilodalton subunits of the PSII core complex. These observations indicate that additional mechanisms may also exist to help optimize the interception of quanta during rapid changes in illumination conditions.     

Determination of Ion Content and Ion Fluxes in the Halotolerant Alga Dunaliella salina

A method to determine intracellular cation contents in Dunaliella by separation on cation-exchange minicolumns is described. The separation efficiency of cells from extracellular cations is over 99.9%; the procedure causes no apparent perturbation to the cells and can be applied to measure both fluxes and internal content of any desired cation. Using this technique it is demonstrated that the intracellular averaged Na⁺, K⁺, and Ca²⁺ concentrations in Dunaliella salina cultured at 1 to 4 molar NaCl, 5 millimolar K⁺, and 0.3 millimolar Ca²⁺ are 20 to 100 millimolar, 150 to 250 millimolar, and 1 to 3 millimolar, respectively. The intracellular K⁺ concentration is maintained constant over a wide range of media K⁺ concentrations (0.5-10 millimolar), leading to a ratio of K⁺ in the cells to K⁺ in the medium of 10 to 1,000. Severe limitation of external K⁺, induces loss of K⁺ and increase in Na⁺ inside the cells. The results suggest that Dunaliella cells possess efficient mechanisms to eliminate Na⁺ and accumulate K⁺ and that intracellular Na⁺ and K⁺ concentrations are carefully regulated. The contribution of the intracellular Na⁺ and K⁺ salts to the total osmotic pressure of cells grown at 1 to 4 molar NaCl, is 5 to 20%.     

Transcriptome Profiling of the Salt-Stress Response in the Halophytic Green Alga Dunaliella salina

Plant Molecular Biology Reporter - Due to the hypersaline environment cell of Dunaliella salina can change its morphology, growth, and pigment for adapting to the stress. Despite the fact D. salina...     

Partial Characterization of K and Ca Uptake Systems in the Halotolerant Alga Dunaliella salina

The uptake of K⁺ and Ca²⁺ in Dunaliella salina is mediated by two distinct carriers: a K⁺ carrier with a high selectivity against Na⁺, Li⁺, and choline⁺ but not towards Rb⁺, K⁺, Cs⁺, or NH₄⁺, and a Ca²⁺ carrier with a high selectivity against Mg²⁺. The latter is specifically blocked by La³⁺ and by Cd²⁺. Apparent K_m values for K⁺ and Ca²⁺ uptake are 2.5 and 0.8 millimolar, respectively, and their maximal calculated fluxes are 22 and 0.8 nanomoles per square meter per second, respectively. Effects of permeable ions and ionophores on K⁺ and Ca²⁺ uptake suggest that the driving force for their uptake is the transmembrane electrical potential. Inhibitors of ATP production, typical inhibitors of plasma membrane H⁺-ATPases and protonionophores inhibit K⁺ and Ca²⁺ uptake and accelerate K⁺ efflux. The results suggest that an H⁺-ATPase in the cell membrane provides the driving force for K⁺ and Ca²⁺ uptake. Efflux measurements from ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ loaded cells suggest that part of the intracellular K⁺ and most of the intracellular Ca²⁺ is nonexchangeable with the extracellular pool. Correlations between phosphate and K⁺ contents and the effect of phosphate on K⁺ efflux suggest intracellular associations between K⁺ and polyphosphates. On the basis of these results, it is suggested that: (a) K⁺ and Ca²⁺ uptake in D. salina is driven by the transmembrane electrical potential which is generated by the action of an H⁺-ATPase of the plasma membrane. (b) Part of the intracellular K⁺ is associated with polyphosphate bodies, while most of the intracellular Ca²⁺ is accumulated in intracellular organelles in the algal cells.     

Rapid Cytoplasmic Alkalization and Dynamics of Intracellular Compartmentation of Inorganic Phosphate during Adaptation against Salt Stress in a Halotolerant Unicellular Green Alga Dunaliella tertiolecta: 31P-Nuclear Magnetic Resonance Study

By ³¹P-in vivo nuclear magnetic resonance spectroscopy, twointracellular compartments were detected, and their pH valueswere estimated in intact cells of a halotolerant unicellulargreen alga Dunaliella tertiolecta. They were identified as thecytoplasm (pH 7.1) and vacuoles (pH 6.0). Vacuoles were alsovisualized with a fluorescence-differential interference microscope.During the adaptation to the salt stress (NaCl concentrationfrom 0.17 to 1.0 M) where cells rapidly synthesize glycerolas osmoticum, both cytoplasmic and vacuolar pH showed transientincreases to about 8 and 6.5, respectively. Subsequently, cytoplasmicinorganic phosphate level, as well as sugar phosphates and terminalphosphate group levels increased. These drastic changes in chemicalenvironment in the cytoplasm including pH and inorganic phosphateconcentration are discussed to be key factors for osmoregulationthat activate the synthesis and inhibit the breakdown of glycerol. (Received August 22, 1988; Accepted February 6, 1989)     

Na-NMR Studies of the Intracellular Sodium Ion Concentration in the Halotolerant Alga Dunaliella salina

The Intracellular Na⁺ concentration in the halotolerant alga Dunaliella salina was measured in intact cells by ²³Na-NMR spectroscopy, utilizing the dysprosium tripolyphosphate complex as a sodium shift reagent, and was found to be 88 ± 28 millimolar. Intracellular sodium ion content and intracellular volume were the same, within the experimental error, in cells adapted to grow in media containing between 0.1 and 4.0 molar NaCl. These values assume extracellular and intracellular NMR visibilities of the ²³Na nuclei of 100 and 40%, respectively. The relaxation rate of intracellular sodium was enhanced with increasing salinity of the growth medium, in parallel to the intracellular osmosity due to the presence of glycerol, indicating that Na⁺ ions and glycerol are codistribbuted within the cell volume.     

On the Factors Which Determine Massive beta-Carotene Accumulation in the Halotolerant Alga Dunaliella bardawil

Dunaliella bardawil, a β-carotene-accumulating halotolerant alga, has been analyzed for the effect of various growth conditions on its pigment content, and compared with Dunaliella salina, a β-carotene nonaccumulating species. In D. bardawil, increasing light intensity and light period or inhibiting growth by various stress conditions such as nutrient deficiency or high salt concentration caused a decrease in the content of chlorophyll per cell and an increase in the amount of β-carotene per cell. As a result, the β-carotene-to-chlorophyll ratio increased from about 0.4 to 13 grams per gram and the alga changed its visual appearance from green to deep orange. D. salina grown similarly decreased in content of both chlorophyll and β-carotene per cell and the culture turned from green to yellowish. Low chlorophyll-containing cells of D. bardawil or D. salina exhibit very high photosynthetic rates when expressed on a chlorophyll basis (~600 micromoles O₂ evolved per milligram chlorophyll per hour).

Variation of pigment content in D. bardawil by a large variety of environmental agents has been correlated with the integral irradiance received by the algal culture during a division cycle. The higher the integral irradiance per division cycle, the lower the chlorophyll content per cell; the higher the β-carotene content per cell, and therefore the higher the β-carotene-to-chlorophyll ratio. The results are interpreted as indicating a protecting effect of β-carotene against injury by high irradiance under conditions of impairment in chlorophyll content per cell.

     

Plasma Membrane Sterols Are Essential for Sensing Osmotic Changes in the Halotolerant Alga Dunaliella

The halotolerant alga Dunaliella responds to hyperosmotic stress by synthesis of massive amounts of glycerol. The trigger for this osmotic response is the change in cell volume, but the mechanism that senses volume changes is not known. Preincubation of Dunaliella salina with tridemorph, a specific inhibitor of sterol biosynthesis, inhibits glycerol synthesis and volume recovery. The inhibition is associated with suppression of [14C]bicarbonate incorporation into sterols and is correlated with pronounced depletion of plasma membrane sterols. Incubation of sterol-depleted cells with cholesterol hemisuccinate restores the capacity for volume regulation in response to hyperosmotic stress. Tridemorph as well as lovastatin also inhibit volume changes that are induced by high light in Dunaliella bardawil, a species that responds to high light intensity by synthesis of large amounts of [beta]-carotene. These volume changes result from accumulation of glycerol and are associated with de novo synthesis of sterols. The major plasma membrane sterol in D. salina and the high-light-induced sterol in D. bardawil co-migrate with ergosterol on thin-layer chromatography and on reversed-phase high-performance liquid chromatography. These results suggest that the osmosensory mechanism in Dunaliella resides in the plasma membrane, and that sterols have an important role in sensing osmotic changes.     

Amine Accumulation in Acidic Vacuoles Protects the Halotolerant Alga Dunaliella salina Against Alkaline Stress

Amines at alkaline pH induce in cells of the halotolerant alga Dunaliella a transient stress that is manifested by a drop in ATP and an increase of cytoplasmic pH. As much as 300 millimolar NH₄⁺ are taken up by the cells at pH 9. The uptake is not associated with gross changes in volume and is accompanied by K⁺ efflux. Most of the amine is not metabolized, and can be released by external acidification. Recovery of the cells from the amine-induced stress occurs within 30 to 60 minutes and is accompanied by massive swelling of vacuoles and by release of the fluorescent dye atebrin from these vacuoles, suggesting that amines are compartmentalized into acidic vacuoles. The time course of ammonia uptake into Dunaliella cells is biphasic—a rapid influx, associated with cytoplasmic alkalinization, followed by a temperature-dependent slow uptake phase, which is correlated with recovery of cellular ATP and cytoplasmic pH. The dependence of amine uptake on external pH indicates that it diffuses into the cells in the free amine form. Studies with lysed cell preparations, in which vacuoles become exposed but retain their capacity to accumulate amines, indicate that the permeability of the vacuolar membrane to amines is much higher than that of the plasma membrane. The results can be retionalized by assuming that the initial amine accumulation, which leads to rapid vacuolar alkalinization, activates metabolic reactions that further increase the capacity of the vacuoles to sequester most of the amine from the cytoplasm. The results indicate that acidic vacuoles in Dunaliella serve as a high-capacity buffering system for amines, and as a safeguard against cytoplasmic alkalinization and uncoupling of photosynthesis.     

Identification of Low Molecular Mass GTP-Binding Proteins in Membranes of the Halotolerant Alga Dunaliella salina

A family of specific guanine nucleotide-binding proteins in Dunaliella salina was studied. Polypeptides of different subcellular fractions were separated by electrophoresis and transferred to nitrocellulose or Immobilon membranes. Incubation of the transfer blots with [³⁵S]GTPγS or [α-³²P]GTP showed no evidence for GTP-binding proteins in the chloroplast and cytosol fractions. However, two GTP-binding proteins with molecular masses of 28 and 30 kilodaltons were present in the plasma membrane and microsomal fractions. An additional 29 kilodalton GTP-binding protein was detected in the plasma membrane. The mitochondrial fraction contained significant amounts of only the 28 kilodalton GTP-binding protein. Binding of [³²P]GTP to the protein blots was completely prevented by 10 micromolar GTP or guanosine 5′-O-(2-thiodiphosphate) (added in 3 × 10⁴-fold excess), whereas ATP or CTP had no effect on the binding. The 28 kilodalton GTP-binding protein was recognized by polyclonal antibodies to the ras-related YPT1 protein of yeast but not by the anti-ras Y13-259 monoclonal antibody. GTP-binding proteins present in the microsomal fraction could not be solubilized by incubation of microsomes with 1 molar NaCl or 0.2 molar Na₂CO₃, but some GTP-binding activity was solubilized when microsomes were treated with 6 molar urea. These results indicate that D. salina GTP-binding proteins are tightly associated with the membranes. The covalent attachment of fatty acids to these proteins was also investigated. Electrophoresis followed by fluorography of delipidated microsomal proteins extracted from [³H]myristic acid-labeled cells showed an intense labeling of a 28 kilodalton protein. We conclude that D. salina contains proteins resembling the ras-related proteins found in animal cells and higher plants.     

P and C-NMR Studies of the Phosphorus and Carbon Metabolites in the Halotolerant Alga, Dunaliella salina

The intracellular phosphorus and carbon metabolites in the halotolerant alga Dunaliella salina adapted to different salinities were monitored in living cells by ³¹P- and ¹³C-nuclear magnetic resonance (NMR) spectroscopy. The ¹³C-NMR studies showed that the composition of the visible intracellular carbon metabolites other than glycerol is not significantly affected by the salinity of the growth medium. The T₁ relaxation rates of the ¹³C-glycerol signals in intact cells were enhanced with increasing salinity of the growth medium, in parallel to the expected increase in the intracellular viscosity due to the increase in intracellular glycerol. The ³¹P-NMR studies showed that cells adapted to the various salinities contained inorganic phosphate, phosphomonoesters, high energy phosphate compounds, and long chain polyphosphates. In addition, cells grown in media containing up to 1 molar NaCl contained tripolyphosphates. The tripolyphosphate content was also controlled by the availability of inorganic phosphate during cell growth. Phosphate-depleted D. salina contained no detectable tripolyphosphate signal. Excess phosphate, however, did not result in the appearance of tripolyphosphate in ³¹P-NMR spectra of cells adapted to high (>1.5 molar NaCl) salinites.     

Physiological responses of Dunaliella salina and Dunaliella tertiolecta to copper toxicity

Species differences in heavy metal tolerance were investigated by comparing the responses of Dunaliella tertiolecta and Dunaliella salina to elevated concentrations of CuCl2. Although both species showed reduced cell number ml(-1) of algal culture, D. salina was more affected by increase in CuCl2. This reflects higher sensitivity of D. salina to CuCl2 compared to D. tertiolecta. Total chlorophyll in terms of microg ml(-1) was higher in D. tertiolecta at all tested CuCl2 levels, but in terms of microg cell(-1) no significant difference was observed between the two species. Total carotenoids in microg cell(-1) increased with increase in CuCl2 in both species and it was about five times higher in D. salina at all CuCl2 concentrations. While both species showed significant increase in lipid peroxidation at elevated CuCl2, the malondialdehyde content of D. salina cells was about three times higher at most CuCl2 concentrations. Although ascorbate peroxidase (APX) activity increased with increase in CuCl2 levels in both species, higher activity was observed in D. tertiolecta at all tested CuCl2 concentrations. Cu content of D. salina cells was higher than D. tertiolecta which may be due to larger volume of D. salina cells. In conclusion, since hydroxyl radical (HO*) produced from H2O2 by Cu2+ (Haber-Weiss cycle) is involved in lipid peroxidation, higher ascorbate peroxidase activity in D. tertiolecta may partly account for lower sensitivity of this species to CuCl2 compared to D. salina.