Uri Pick

Liposomes with a large trapping capacity prepared by freezing and thawing of sonicated phospholipid mixtures

Abstract A reconstitution procedure for a glucose-transporting protein based on a rapid freezing of sonicated phospholipid mixtures with the solubilized protein followed by thawing and a brief sonication was described by Kasahara and Hinkle (1977, J. Biol. Chem.252, 7384–7390). In this paper we demonstrate that the liposomes prepared by this simple procedure referred to as the freezing-thawing-sonication procedure have a specific trapping volume of 10 μl H2O/mg phospholipid and a maximal trapping efficiency of 25–30% of the solution including ions, small organic molecules, or proteins. Formation of large liposomes by this technique which probably results from fusion of sonicated liposome is strongly inhibited by increasing the ionic strength of the medium, by sucrose, and by increasing the liposome concentration. The freezing-thawing-sonication procedure is effective with a crude phospholipid extract obtained from soybeans or with mixtures of phosphatidylcholine with either a negatively charged phospholipid (phosphatidylserine) or a positively charged lipid (cetyltrimethylammonium bromide) but not with pure phosphatidylcholine liposomes. Based on these findings, we suggest that water molecules have to crystallize on the charged phospholipid interface during the rapid freezing in order to allow the fusion process to take place. Liposomes prepared by the freezing-thawing-sonication procedure have a low permeability to protons, cations, and anions which makes them a useful system for studies of ion transport. We give a few examples of ionophore-mediated ion transport reactions which give rise to accumulation of protons and Ca ions inside the lipsomes.

Enhanced Photosynthesis and Redox Energy Production Contribute to Salinity Tolerance in Dunaliella as Revealed by Homology-Based Proteomics

Salinity is a major limiting factor for the proliferation of plants and inhibits central metabolic activities such as photosynthesis. The halotolerant green alga Dunaliella can adapt to hypersaline environments and is considered a model photosynthetic organism for salinity tolerance. To clarify the molecular basis for salinity tolerance, a proteomic approach has been applied for identification of salt-induced proteins in Dunaliella. Seventy-six salt-induced proteins were selected from two-dimensional gel separations of different subcellular fractions and analyzed by mass spectrometry (MS). Application of nanoelectrospray mass spectrometry, combined with sequence-similarity database-searching algorithms, MS BLAST and MultiTag, enabled identification of 80% of the salt-induced proteins. Salinity stress up-regulated key enzymes in the Calvin cycle, starch mobilization, and redox energy production; regulatory factors in protein biosynthesis and degradation; and a homolog of a bacterial Na+-redox transporters. The results indicate that Dunaliella responds to high salinity by enhancement of photosynthetic CO2 assimilation and by diversion of carbon and energy resources for synthesis of glycerol, the osmotic element in Dunaliella. The ability of Dunaliella to enhance photosynthetic activity at high salinity is remarkable because, in most plants and cyanobacteria, salt stress inhibits photosynthesis. The results demonstrated the power of MS BLAST searches for the identification of proteins in organisms whose genomes are not known and paved the way for dissecting molecular mechanisms of salinity tolerance in algae and higher plants.

Indications for an oligomeric structure and for conformational changes in sarcoplasmic reticulum Ca2+-ATPase labelled selectively with fluorescein

Fluorescein isothiocyanate is a highly specific inhibitor of the Ca2+-ATPase from sarcoplasmic reticulum. The Ca2+ pumping is inhibited completely at a fluorescein isothiocyanate concentration half that of the ATPase protein, indicating that the protein is at least a dimer. ATP protected specifically against fluorescein isothiocyanate inhibition, indicating that fluorescein isothiocyanate may react at the nucleotide binding site of the ATPase (probably with a reactive lysine residue). The fluorescein is incorporated almost exclusively into the 105 kdalton catalytic polypeptide of the ATPase and digestion by trypsin gives rise to a fluorescein-labelled 45 kdalton fragment. Conformational changes induced by addition of Ca can be studied conveniently with the fluorescein-labelled ATPase.

Salt-induced Changes in the Plasma Membrane Proteome of the Halotolerant Alga Dunaliella salina as Revealed by Blue Native Gel Electrophoresis and Nano-LC-MS/MS Analysis

The halotolerant alga Dunaliella salina is a recognized model photosynthetic organism for studying plant adaptation to high salinity. The adaptation mechanisms involve major changes in the proteome composition associated with energy metabolism and carbon and iron acquisition. To clarify the molecular basis for the remarkable resistance to high salt, we performed a comprehensive proteomics analysis of the plasma membrane. Plasma membrane proteins were recognized by tagging intact cells with a membrane-impermeable biotin derivative. Proteins were resolved by two-dimensional blue native/SDS-PAGE and identified by nano-LC-MS/MS. Of 55 identified proteins, about 60% were integral membrane or membrane-associated proteins. We identified novel surface coat proteins, lipid-metabolizing enzymes, a new family of membrane proteins of unknown function, ion transporters, small GTP-binding proteins, and heat shock proteins. The abundance of 20 protein spots increased and that of two protein spots decreased under high salt. The major salt-regulated proteins were implicated in protein and membrane structure stabilization and within signal transduction pathways. The migration profiles of native protein complexes on blue native gels revealed oligomerization or co-migration of major surface-exposed proteins, which may indicate mechanisms of stabilization at high salinity.

A Salt-resistant Plasma Membrane Carbonic Anhydrase Is Induced by Salt in Dunaliella salina

The mechanisms allowing proliferation of the unicellular green alga Dunaliella salina in up to saturating NaCl concentrations are only partially understood at present. Previously, the level of a plasma membrane Mr 60,000 protein, p60, was found to increase with rising external salinities. Based on cDNA cloning and enzymatic assays, it is now shown that p60 is an internally duplicated carbonic anhydrase, with each repeat homologous to animal and Chlamydomonas reinhardtii carbonic anhydrases, but exceptional in the excess of acidic over basic residues. Increasing salinities, alkaline shift, or removal of bicarbonate induced in D. salina parallel increases in the levels of p60, its mRNA, and external carbonic anhydrase activity. Moreover, purified p60 exhibited carbonic anhydrase activity comparable to other carbonic anhydrases. A p60-enriched soluble preparation showed maximal carbonic anhydrase activity at ∼1.0 M NaCl and retained considerable activity at higher salt concentrations. In contrast, a similar preparation from C. reinhardtii was ∼90% inhibited in 0.6 M NaCl. These results identified p60 as a structurally novel carbonic anhydrase transcriptionally regulated by CO2 availability and exhibiting halophilic-like characteristics. This enzyme is potentially suited to optimize CO2 uptake by cells growing in hypersaline media.

Modification of the ATP binding site of the Ca2+‐ATPase from sarcoplasmic reticulum by fluorescein isothiocyanate

Fluorescein isothiocyanate, generally used as a nonspecific fluorescent probe for labeling of proteins, was demonstrated to be an efficient inhibitor of the Na’, K’-ATPase [l] and of the Ca2+-ATPase from sarcoplasmic reticulum [2]. Incubation of the Ca”-ATPase from sarcoplasmic reticulum with PM levels of FITC at alkaline pH led to complete inhibition of the activity within a few minutes. Binding of 1 fluorescein molecule/2 Ca’+-ATPase polypeptides (M, 105 000) resulted in complete inhibition of the activity which may suggest that the active form of the enzyme is at least a dimer. ATP effectively protected against FITC inhibition both the Na+, K’-ATPase and the Ca2+ATPase [ 1,2]. Taken together these results may suggest that FITC is a selective affinity probe for the ATP binding site probably labeling a lysine group at or close to the ATP binding site in both ATPases. Here, we demonstrate that FITC specifically blocks the ATP binding site of the Ca2+-ATPase from sarcoplasmic reticulum and does not prevent the phosphorylation of the enzyme by Pi or Ca2+-uptake with acetyl phosphate as a substrate. The implications of these findings for the role of the regulatory ATP binding site are discussed. The usefulness of this probe to follow interconversions between phosphorylated and non-phosphorylated conformational states of the Ca2*-ATPase is described in [3].

A Structurally Novel Transferrin-like Protein Accumulates in the Plasma Membrane of the Unicellular Green Alga Dunaliella salina Grown in High Salinities

The alga Dunaliella salina is outstanding is its ability to withstand extremely high salinities. To uncover mechanisms underlying salt tolerance, a search was carried out for salt-induced proteins. The level of a plasma membrane 150-kDa protein, p150, was found to increase with rising external salinity (Sadka, A., Himmelhoch, S., and Zamir, A. (1991) Plant Physiol. 95, 822-831). Based on its cDNA-deduced sequence, p150 belongs to the transferrin family of proteins so far identified only in animals. This, to our best knowledge, is the first demonstration of a transferrin-like protein in a photosynthetic organism. Unlike animal transferrins, p150 contains three, rather than two, internal repeats and a COOH-terminal extension including an acidic amino acid cluster. In intact cells p150 is degraded by Pronase, indicating that the protein is extracellularly exposed. The relationship of p150 to iron uptake is supported by the induction of the protein in iron-deficient media and by its radioactive labeling in cells grown with 59Fe. Accumulation of p150 is transcriptionally regulated. It is proposed that p150 acts in iron uptake other than by receptor-mediated endocytosis and that its induction permits the cells to overcome a possible limitation in iron availability under high salinities.

The dependence of photophosphorylation in chloroplasts on ΔpH and external pH

The rate of photophosphorylation in broken chloroplasts is strongly dependent on medium pH with an optimum around 8.3 [ 11. Several factors may contribute to this pH dependence: (a) the pH dependence of the phosphorylating enzyme complex itself; (b) the pH dependence of electron transport which drives the phosphorylation, and (c) since it has been demonstrated that in chloroplasts a correlation exists between the size of the pH gradient maintained across the thylakoid membrane (ApH) and the efficiency of energy conversion [2,4], it would be expected that the rate of ATP formation will depend on the magnitude of ApH, which itself is strongly pH dependent [3,1 l] . It was previously shown [S] that the rate of electron transport was mostly dependent on internal pH, but also on ApH and external pH. If the driving force for phosphorylation is provided by the electrochemical gradient of protons across the chloroplasts membrane [6] , one should expect that the membrane potential could also act as an energy source for phosphorylation. Evidence was presented recently [7,8] which indicated that under the condition of limiting ApH, an externally induced diffusion potential could indeed drive phosphorylation. Nevertheless, we have previously concluded that during light-dependent phosphorylation, the size of the membrane potential was too small to play any significant role in energy conversion [3]. It has been

Iron Uptake by the Halotolerant Alga Dunaliella Is Mediated by a Plasma Membrane Transferrin

A 150-kDa transferrin-like protein (Ttf) is associated with the plasma membrane of the halotolerant unicellular alga Dunaliella salina (Fisher, M., Gokhman, I., Pick, U., and Zamir, A. (1997) J. Biol. Chem. 272, 1565–1570). The Ttf level rises with medium salinity or upon iron depletion. Evidence that Ttf is involved in iron uptake by Dunaliellais presented here. Algal iron uptake exhibits characteristics resembling those of animal transferrins: high specificity and affinity for Fe3+ ions, strict dependence on carbonate/bicarbonate ions, and very low activity in acidic pH. Reducing the level of Ttf by mild proteolysis of whole cells is accompanied by lowered uptake activity. Conversely, accumulation of high levels of Ttf is correlated with an enhancement of iron uptake. Kinetically, iron uptake consists of two steps: an energy-independent binding of iron to the cell surface and an energy-dependent internalization. Salinities as high as 3.5 m NaCl do not inhibit iron uptake or decrease the apparent affinity for Fe3+ ions, implying that Ttf activity is not affected by high salt. These results indicate that transferrins, hitherto identified only in animals, are present and function in iron transport also in plant systems.

Are active oxygen species involved in induction of β-carotene in Dunaliella bardawil?

The purpose of this work was to test whether induction of massive β-carotene synthesis in the alga Dunaliella bardawil is triggered by oxygen radicals. The following results were obtained: (i) The induction of β-carotene synthesis is preceded by a lag period of about 4 h during which the cells swell and photosynthesis is partially inhibited, (ii) Addition of promoters of oxygen radicals or of azide (an inhibitor of catalase and superoxide dismutase) during the induction period, under conditions which are suboptimal for massive β-carotene accumulation, greatly enhances β-carotene synthesis, photodegradation of chlorophyll and inhibition of photosynthesis, (iii) High irradiance, which induces massive β-carotene accumulation, also induces a high catalase activity. It is suggested that photosynthetically produced oxygen radicals are involved in triggering massive β-carotene accumulation in D. bardawil.