Salt induction of fatty acid elongase and membrane lipid modifications in the extreme halotolerant alga Dunaliella salina

In studies of the outstanding salt tolerance of the unicellular green alga Dunaliella salina, we isolated a cDNA for a salt-inducible mRNA encoding a protein homologous to plant beta-ketoacyl-coenzyme A (CoA) synthases (Kcs). These microsomal enzymes catalyze the condensation of malonyl-CoA with acyl-CoA, the first and rate-limiting step in fatty acid elongation. Kcs activity, localized to a D. salina microsomal fraction, increased in cells transferred from 0.5 to 3.5 M NaCl, as did the level of the kcs mRNA. The function of the kcs gene product was directly demonstrated by the condensing activity exhibited by Escherichia coli cells expressing the kcs cDNA. The effect of salinity on kcs expression in D. salina suggested the possibility that salt adaptation entailed modifications in the fatty acid composition of algal membranes. Lipid analyses indicated that microsomes, but not plasma membranes or thylakoids, from cells grown in 3.5 M NaCl contained a considerably higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids compared with cells grown in 0.5 M salt. Thus, the salt-inducible Kcs, jointly with fatty acid desaturases, may play a role in adapting intracellular membrane compartments to function in the high internal glycerol concentrations balancing the external osmotic pressure.     

Temperature-regulated expression of a glycine-rich RNA-binding protein in the halotolerant alga Dunaliella salina

Acclimation of the halotolerant alga Dunaliella salina to low temperature induced the accumulation of a 12.4 kDa protein (DsGRP-1) and reduction of a 13.1 kDa protein (DsGRP-2). DsGRP-1 and DsGRP-2 are boiling-stable proteins that are localised in the cytoplasm, as revealed by sub-cellular fractionation and by immuno-localisation. The proteins were partially purified and their corresponding genes were cloned. The predicted sequences are homologous to Glycine-Rich RNA-binding Proteins (GRPs) from plants and cyanobacteria. The nucleotide sequences of grp1 and grp2 differ in a short insert encoding 9 amino acids in the glycine-rich domain of DsGRP-2. grp2 contains a single intron at position 179 indicating that DsGRP-1 and DsGRP-2 are not derived from alternative splicing of a common gene. The level of grp mRNA increased at 7 degrees C and was rapidly depressed at 24 degrees C. Analysis of binding to ribonucleotide homopolymers revealed that DsGRP-1 and DsGRP-2 bind preferentially to poly-G and to poly-U indicating that they are RNA-binding proteins. It is proposed that DsGRP-1 and DsGRP-2 are encoded by distinct genes which are differentially regulated by temperature.     

Analysis of Streptomyces coelicolor membrane proteome using two-dimensional native/native and native/sodium dodecyl sulfate gel electrophoresis

Analysis of the oligomeric state of a protein may provide insights into its physiological functions. Because membrane proteins are considered to be the workhorses of energy generation and polypeptide and nutrient transportation, in this study we characterized the membrane-associated proteome of Streptomyces coelicolor by two-dimensional (2D) blue native/sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), high-resolution clear native/native PAGE, and native/SDS–PAGE. A total of 77 proteins were identified, and 20 proteins belonging to 15 complexes were characterized. Moreover, the resolution of high-resolution clear native/SDS–PAGE is much higher than that of blue native/SDS–PAGE. OBP (SCO5477) and BldKB (SCO5113) were identified as the main protein spots from the membrane fractions of S. coelicolor M145, suggesting that these two proteins are involved in extracellular peptide transportation. These two transporters exhibited multiple oligomeric states in the native PAGE system, which may suggest their multiple physiological functions in the development of S. coelicolor.     

Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina

Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the 14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca²⁺-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein gene is cell cycle-dependent.     

NaCl-induced phosphorylation of light harvesting chlorophyll a/b proteins in thylakoid membranes from the halotolerant green alga, Dunaliella salina

Light could induce phosphorylation of light harvesting chlorophyll a/b binding proteins (LHCII) in Dunaliella salina and spinach thylakoid membranes. We found that neither phosphorylation was affected by glycerol, whereas treatment with NaCl significantly enhanced light-induced LHCII phosphorylation in D. salina thylakoid membranes and inhibited that in spinach. Furthermore, even in the absence of light, NaCl and several other salts induced LHCII phosphorylation in D. salina thylakoid membranes, but not in spinach thylakoid membranes. In addition, hypertonic shock induced LHCII phosphorylation in intact D. salina under dark conditions and cells adapted to different NaCl concentrations exhibited similar LHCII phosphorylation levels. Taken together, these results show for the first time that while LHCII phosphorylation of D. salina thylakoid membranes resembles that of spinach thylakoid membranes in terms of light-mediated control, the two differ with respect to NaCl sensitivity under light and dark conditions.     

Characterization of the human skeletal muscle proteome by one-dimensional gel electrophoresis and HPLC-ESI-MS/MS

Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including 55 subunits of the respiratory complexes I-V. Moreover, a number of enzymes involved in endocrine and metabolic signaling pathways as well as calcium homeostasis were identified. These results provide the most comprehensive characterization of the human skeletal muscle proteome to date. These data hold promise for future global assessment of quantitative changes in the muscle proteome of patients affected by disorders involving skeletal muscle.     

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.     

Biphasic changes in the level and composition of Dunaliella salina plasma membrane diacylglycerols following hypoosmotic shock.

Hypoosmotic shock has been shown to trigger an immediate and selective increase of plasma membrane diacylglycerols (DAG) in the green alga Dunaliella salina, coinciding with an approximately equivalent loss of phosphatidylinositol 4,5-bisphosphate from this membrane [Ha, K.S., & Thompson, G.A., Jr. (1991) Plant Physiol. 97, 921-927]. Following a slight decline in amount, DAG levels of the plasma membrane resumed their rise by 2 min after the shock and by 40 min had achieved a maximum concentration equivalent to 230% of DAG levels in unstressed cells. This second, more sustained increase of plasma membrane DAG was matched by a DAG increase in the microsome-enriched cytoplasmic membrane fraction, commencing at 2 min and peaking at 140% of control values. The changing pattern of DAG molecular species produced in the plasma membrane during the early phases of hypoosmotic stress was compatible with their derivation from phospholipase C hydrolysis of inositol phospholipids and phosphatidylcholine. From 8 min following hypoosmotic shock, as relatively larger scale DAG accumulations developed in the cytoplasmic membranes, the molecular species composition changed to reflect a marked increase in de novo synthesis of sn-1-oleoyl, sn-2-palmitoylglycerol, and dioleoylglycerol. The former molecular species appears to be synthesized in the chloroplast while the latter is produced in the endoplasmic reticulum. The radioisotope labeling data with Na2(14)CO3 confirmed that the biphasic formation of DAG triggered by hypoosmotic shock culminates in a large-scale de novo synthesis of DAG. This is the first clear evidence for de novo synthesis as a source of DAG following PIP2-mediated signaling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and nucleotide sequence analysis of genes coding for the major chlorophyll-binding protein of the moss Physcomitrella patens and the halotolerant alga Dunaliella salina

Two clones have been isolated from a genomic library of the moss Physcomitrella patens and a cDNA library of the halotolerant green alga Dunaliella salina. The isolates contain genes coding for the major light-harvesting chlorophyll-a/b-binding protein (CAB) in the photosystem II (PSII) light-harvesting complex (LHCII). The 2544-bp insert of the moss genomic clone contains the complete CAB-coding region and 5' and 3' flanking sequences. The coding region contains an intron of 359 bp which is spanned by a pair of 9-bp perfect direct repeats. There are two CCAAT boxes and five enhancer-like elements related to (G)TGGTTTAAA(G) (Weiher et al., 1983) residing in the intron. Comparisons of the moss cab gene with sequences of light-inducible genes of higher plants reveal homologous and repeated sequences similar to the enhancer element in the 5' region upstream from the TATA and CCAAT boxes thought to be responsive to light inducibility. The 1256-bp algal cDNA contains the complete CAB-coding sequence, a 170-bp 5'-nontranslated region, and a 264-bp 3'-nontranslated region. While the overall homology in the nontranslated regions is low between the cab gene of the moss and that of the alga, the 3'-nontranslated regions of the two contain some sequences that are conserved among the cab genes in higher plants. The deduced amino acid sequences of these two clones are highly conserved except for the N-terminal region. Their hydropathic plots are very similar and both possess three hydrophobic segments that are likely alpha-helical transmembrane segments. The proposed CAB transit peptide sequence of the alga is divergent from that of the moss or higher plants, suggesting that they may have evolved from different origins. Southern blot analysis shows that the cab genes in the moss and the alga, as in higher plants, are encoded by a number of homologous genes constituting a multigene family.     

Phosphorus metabolism and intracellular pH in the halotolerant alga Dunaliella parva studied by 31P-NMR

The metabolism of the green unicellular halotolerant alga Dunaliella parva was studied by means of ³¹P nuclear magnetic resonance spectroscopy. The major soluble phosphate compounds were found to be similar to those in other organisms but two phosphodiesters, glycerophosphorylglycerol and glycerophosphorylcholine, were identified in algal tissue for the first time. Only a single pool of intracellular orthophosphate was observed and the chemical shift of the corresponding resonance was used to monitor the intracellular pH. The cell pH and the orthophosphate content were sensitive both to the oxygenation of the cells and to the illumination of the cell suspension. The intracellular pH was controlled over an external pH range of 6–9, but at pH 5 the cell contents became acidic. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone was observed to uncouple oxidative phosphorylation but it did not equilibrate the pH difference across the cell membrane in experiments conducted at an external pH of 7.8.     

SURFEIT-1 gene analysis and two-dimensional blue native gel electrophoresis in cytochrome c oxidase deficiency

Leigh syndrome, a progressive, often fatal, neurodegenerative disorder, is frequently associated with a deficiency in the activity of cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain. In contrast to NADH:ubiquinone oxidoreductase and succinate dehydrogenase deficiencies, no mutations in nuclear genes encoding COX subunits have been identified thus far. Very recently, however, a Leigh syndrome complementation group has been identified which showed mutations in the SURFEIT-1 (SURF-1) gene. The results of a mutational detection study in 16 new randomly selected COX-deficient patients revealed a new mutation (C688T) in 2 patients and the earlier reported 845delCT mutation in 2 additional patients. In addition, we evaluated the diagnostic value of two-dimensional blue native gel electrophoresis. We show that this technique reveals distinct patterns of both fully and partially assembled COX complexes and is thereby capable of discrimination between COX-deficient SURF-1 and non-SURF-1-mutated patients.     

A high-definition native polyacrylamide gel electrophoresis system for the analysis of membrane complexes

Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN‐) and high resolution clear native (hrCN‐) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine‐ and deoxycholate‐based native (HDN‐) PAGE. We compared the capacity of HDN‐, BN‐ and hrCN‐PAGE to resolve the well‐studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN‐PAGE. The analysis of isolated chloroplast envelope complexes by HDN‐PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN‐PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons.     

Effect of osmotic shock on the redox system in plasma membrane of Dunaliella salina

The unicellular halotolerant alga Dunaliella salina had the ability to oxidize NADH and reduce Fe(CN)6^3-.The redox reactions were to some extent stimulated by slight hyperosmotic shock (2.0mol/L→2.6mol/L NaCl),but markably inhibited by abrupt hyperosmotic shock (2.0mol/L→3.5mol/L NaCl) and hypoosmotic shock (2.0mol/L→1.0mol/L NaCl;2.0mol/L→0.67mol/L NaCl).With the adaptation of algal cells to osmotic shock by accumulating or degraging intracellular grycerol,the plasmalemma redox activities were also restored.The O2 uptake stimulated by NADH could be promoted by FA and SHAM.Hypoosmotic shock increases the basal respiration rate of alga cells,but weakened the stimulating effects of NADH,FA and SHAM on O2 uptake.On the other hand,hyperosmotic shock reduced the basal respiration rate,but relatively enhanced the above effects of NADH,FA and SHAM.H^ extrusion of alga cells was inhibited by NADH and stimulated by Fe(CN)6^3-.Vanadate and DES could inhibit H^ efflux,but had little effect in the presence of NADH and Fe(CN)6^3-.Both hyperand hypoosmotic shock stimulated H^ extrusion.This effect could be totally inhibited by vanadate and DES,but almost unaffected by 8-hydroxyquinoline.It was suggested that H^ -ATPase probably played a more important role in H^ extrusion and osmoregulation under the conditions of osmotic shock.     

Coupling of native liquid phase isoelectrofocusing and blue native polyacrylamide gel electrophoresis: a potent tool for native membrane multiprotein complex separation

In this study, a new 3D native electrophoretic protocol is proposed for an exhaustive separation and identification of membrane proteins. It is based on native liquid phase isoelectrofocusing (N-LP-IEF) of protein complexes in the first dimension, followed by blue native polyacrylamide gel electrophoresis (BN-PAGE) in the second dimension, where both the pI and the molecular masses of protein complexes (2D N-LP-IEF-BN) were used to separate them in their native form. Finally, each single component can be resolved using denaturing electrophoresis (3D N-LP-IEF-BN-SDS-PAGE). The thylakoid membrane of spinach which contains four big protein complexes was chosen as a model for setting up analytical methods suitable for any membrane proteins. The pI-based MicroRotofor has a number of advantages over BN-PAGE: it does not require the addition of any chemicals, and separation of complexes is based on the protein's real physicochemical properties which inevitably change when dye is added. Results were more easily reproduced than with BN, and the pI of each native complex was also determined. Although some fractions still contained comigrating complexes after MicroRotofor, these were subsequently separated by BN for further analysis. Thus, highly hydrophobic complexes, such as ATP-synthetas and Cyt b6/f, were separated in native form as were various complexes of LHCII trimers, which have different pI but similar molecular masses. SDS-PAGE revealed almost all the subunits from the four photosynthetic complexes, indicating that by using 3D N-LP-IEF-BN-SDS-PAGE it is possible to achieve a greater degree of component identification than with 2D BN-SDS-PAGE.     

Na+-transporting ATPase in the plasma membrane of halotolerant microalga Dunaliella maritima operates as a Na+ uniporter

A fraction of inside-out membrane vesicles enriched in plasma membranes (PM) was isolated from Dunaliella maritima cells. Attempts were made to reveal ATP-driven Na⁺-dependent H⁺ efflux from the PM vesicles to external medium, as detected by alkalization of the vesicle lumen. In parallel experiments, ATP-dependent Na⁺ uptake and electric potential generation in PM vesicles were investigated. The alkalization of the vesicle lumen was monitored with an impermeant pH-sensitive optical probe pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid), which was loaded into vesicles during the isolation procedure. Sodium uptake was measured with ²²Na⁺ radioactive label. The generation of electric potential in PM vesicles (positive inside) was recorded with a voltage-sensitive probe oxonol VI. Appreciable Na⁺-and ATP-dependent alkalization of vesicle lumen was only observed in the presence of a protonophore CCCP (carbonyl cyanide-chlorophenylhydrazone). In parallel experiments, CCCP accelerated the ATP-dependent ²²Na⁺ uptake and abolished the electric potential generated by the Na⁺-ATPase at the vesicle membrane. A permeant anion NO^? ₃ accelerated ATP-dependent ²²Na⁺ uptake and promoted dissipation of the electric potential like CCCP did. At the same time, NO^? ₃ inhibited the ATP-and Na⁺-dependent alkalization of the vesicle lumen. The results clearly show that the ATP-and Na⁺-dependent H⁺ efflux from PM vesicles of D. maritima is driven by the electric potential generated at the vesicle membrane by the Na⁺-ATPase. Hence, the Na⁺-transporting ATPase of D. maritima carries only one ion species, i.e., Na⁺. Proton is not involved as a counter-ion in the catalytic cycle of this enzyme.     

Yeast plasma membrane ghosts. An analysis of proteins by two-dimensional gel electrophoresis

We have examined yeast cell ghost preparations to assess their value in obtaining plasma membrane proteins. Ghosts prepared by two methods involving stabilization of spheroplast envelopes had similar protein patterns by two-dimensional gel electrophoresis, and approximately 200 proteins were resolved. Spheroplasts were lactoperoxidase iodinated, and recovery of label in ghost preparations was greater than 60%. Spheroplasts appeared to be impermeable to the lactoperoxidase reagents as judged by an examination of two-dimensional gel electrophoretic patterns of ghost proteins that had been iodinated in spheroplasts or in unsealed ghosts. Spheroplasts were also impermeable to pronase proteases. Surface iodination and surface proteolysis allowed us to identify exposed ghost proteins; the major ghost glycoprotein was exposed in spheroplasts.Two-dimensional patterns of ghost proteins were not heavily contaminated (?25% of all proteins) by proteins present in soluble or promitochondrial fractions, and estimates of surface label and total cell protein recovery suggested that the ghost fraction represents a cell envelope enrichment of 8–10 fold over whole cells.Resolution of ghost proteins by two-dimensional gel electrophoresis appears to be a powerful aid toward identifying membrane proteins.     

The respiratory inhibitor antimycin A specifically binds Fe(III) ions and mediates utilization of iron by the halotolerant alga Dunaliella salina (Chlorophyta)

It is demonstrated that Antimycin A (AA), a respiratory inhibitor produced by Streptomyces bacteria, forms lipophylic complexes with Fe(III) ions. Spectroscopic titration indicates that Fe(III) ions interact with 2AA molecules. At growth-limiting Fe concentrations, AA mediates Fe uptake and promotes growth and chlorophyll synthesis better than other Fe chelators in the halotolerant alga Dunaliella salina. It is proposed that AA enhances Fe bioavailability in hypersaline solutions by formation of lipophylic Fe-AA complexes which are taken-up and utilized by the algae. The results suggest that the respiratory inhibitor AA can affect Fe metabolism in microorganisms.