Adriana Katz

ACCUMULATION OF β‐CAROTENE IN HALOTOLERANT ALGE: PURIFICATION AND CHARACTERIZATION OF β‐CAROTENE‐RICH GLOBULES FROM DUNALIELLA BARDAWIL (CHLOROPHYCEAE)1

Dunaliella bardawil Ben‐Amotz & Avron, but not most other Dunaliella species, has a unique property of being able to accumulate, in addition to glycerol, large amounts of β‐carotene when cultivated under appropriate conditions. These include high light intensity, a high sodium chloride concentration, nitrate deficiency and extreme temperatures. Under conditions of maximal carotene accumulation D. bardawil contains at least 8% of its dry weight as β‐carotene while D. salina grown under similar conditions contains only about 0.3%. Electron micrographs of D. bardawil grown under conditions of high β‐carotene accumulation show many β‐carotene containing globules located in the interthylakoid spaces of the chloroplast. The same algae grown under conditions where β‐carotene does not accumulate, contain few to no β‐carotene globules. The β‐carotene‐rich globules were released from the algae into an aqueous medium by a two‐stage osmotic shock technique and further purified by centrifugal ion on 10% sucrose. The isolated purified globules were shown by electron microscopy to be free of significant contamination and composed of membrane‐free osmiophilic droplets with an average diameter of 150 nm. Reversed phase high performance liquid chromatography of a total pigment extract of the cells revealed the presence of β‐carotene as the major pigment, together with chlorophylls a and b, α‐carotene and the xanthophylls lutein, neoxauthin and zeaxanthin. β‐Carotene accounted for essentially all the pigment in the purified globules. Analysis of the algal and globule β‐carotene fractions by HPLC showed that the β‐carotene was composed of approximately equal amounts of all‐trans β‐carotene and of its 9‐cis isomer. Intact D. bardawil cells contained on a dry weight basis about 30% glycerol, 30% protein, 18% lipid, 11% carbohydrate, 9%β‐carotene and 1% chlorophyll. The β‐carotene globules were composed of practically only neutral lipids, more than half of which was β‐carotene. It is suggested that the β‐carotene globules may serve to protect D. bardawil against injury by the high intensity irradiation to which this alga is usually exposed in nature.

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.

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.

Selectivity of Digitalis Glycosides for Isoforms of Human Na,K-ATPase

There are four isoforms of the α subunit (α1–4) and three isoforms of the β subunit (β1–3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. α2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An α2-selective cardiac glycoside could provide important insights into physiological and pharmacological properties of α2. The isoform selectivity of a large number of cardiac glycosides has been assessed utilizing α1β1, α2β1, and α3β1 isoforms of human Na,K-ATPase expressed in Pichia pastoris and the purified detergent-soluble isoform proteins. Binding affinities of the digitalis glycosides, digoxin, β-methyl digoxin, and digitoxin show moderate but highly significant selectivity (up to 4-fold) for α2/α3 over α1 (KD α1 > α2 = α3). By contrast, ouabain shows moderate selectivity (≈2.5-fold) for α1 over α2 (KD α1 ≤ α3 < α2). Binding affinities for the three isoforms of digoxigenin, digitoxigenin, and all other aglycones tested are indistinguishable (KD α1 = α3 = α2), showing that the sugar determines isoform selectivity. Selectivity patterns for inhibition of Na,K-ATPase activity of the purified isoform proteins are consistent with binding selectivities, modified somewhat by different affinities of K+ ions for antagonizing cardiac glycoside binding on the three isoforms. The mechanistic insight on the role of the sugars is strongly supported by a recent structure of Na,K-ATPase with bound ouabain, which implies that aglycones of cardiac glycosides cannot discriminate between isoforms. In conclusion, several digitalis glycosides, but not ouabain, are moderately α2-selective. This supports a major role of α2 in cardiac contraction and cardiotonic effects of digitalis glycosides.

Characterization of major lipid droplet proteins from Dunaliella.

Many green algal species can accumulate large amounts of triacylglycerides (TAG) under nutrient deprivation, making them a potential source for production of biodiesel. TAG are organized in cytoplasmic lipid bodies, which contain a major lipid droplet protein termed MLDP. Green algae MLDP differ in sequence from plant oleosins and from animal perilipins, and their structure and function are not clear. In this study, we describe the isolation of MLDP from three species of the extreme halotolerant green algae Dunaliella. Sequence alignment with other green algae MLDP proteins identified a conserved 4-proline domain that may be considered as a signature domain of Volvocales green algae MLDP. Gold immunolabeling localized MLDP at the surface of lipid droplets in D. salina. The induction of MLDP by nitrogen deprivation is kinetically correlated with TAG accumulation, and inhibition of TAG biosynthesis impairs MLDP accumulation suggesting that MLDP induction is co-regulated with TAG accumulation. These results can lead to a better understanding of the structure and function of Volvocales green algae MLDP proteins.

Isolation and Characterization of a Protein Associated with Carotene Globules in the Alga Dunaliella bardawil

The halotolerant alga Dunaliella bardawil accumulates very large amounts of [beta]-carotene when exposed to high light intensity. The accumulated [beta]-carotene is concentrated in small, oily globules within the chloroplast and has been suggested to protect the alga against photodamage by high irradiation (A. Ben-Amotz, A. Katz, M. Avron [1982] J Phycol 18:529–537;A. Ben-Amotz, M. Avron [1983] Plant Physiol 72: 593–597; A. Ben-Amotz, A. Shaish, M. Avron [1989] Plant Physiol 91: 1040–1043). A 38-kD protein was identified and purified from [beta]-carotene globules and was designated carotene globule protein (Cgp). Induction of Cgp occurs in parallel with [beta]-carotene accumulation in D. bardawil grown under different inductive conditions. Cgp is overproduced in a constitutive mutant strain that overproduces [beta]-carotene and is not detected in Dunaliella salina, a species that does not accumulate [beta]-carotene. Cgp production was not suppressed by norflurazon, an inhibitor of [beta]-carotene synthesis that leads to accumulation of the carotenoid precursor phytoene. Immunogold-labeling analysis by electron microscopy demonstrates that the protein is localized at the periphery of the globules. Proteolytic cleavage by trypsin enhances the coalescence and destruction of the globules, in parallel with Cgp disappearance. It is suggested that the function of Cgp is to stabilize the structure of the globules within the chloroplast.

Characterization and reconstitution of the Na+/H+ antiporter from the plasma membrane of the halotolerant alga Dunaliella.

Na+/H+ exchange activity in plasma membrane preparations isolated from the unicellular halotolerant alga, Dunaliella salina, is shown to be competitively inhibited by amiloride or Li+, with Ki values of 25 and 30 microM, respectively. The activity can be followed by either the sodium-dependent change in transvesicular delta pH, as monitored by absorbance changes of Acridine orange, or by the delta pH-dependent uptake of 22Na+ into the intravesicular space. The activity was solubilized, by extraction with Triton, and reconstituted into active proteoliposomes. The activity of the reconstituted proteoliposomes was strongly stimulated by the presence of valinomycin and KCl, suggesting that the exchanger is electrogenic, presumably exchanging more than one proton for each Na+ ion. Partial purification of the Triton-extracted exchanger was obtained by fractionation on a DEAE-cellulose column.

A Multicopper Ferroxidase Involved in Iron Binding to Transferrins in Dunaliella salina Plasma Membranes

The halotolerant alga Dunaliella salina is unique among plants in that it utilizes a transferrin (TTf) to mediate iron acquisition (Fisher, M., Zamir, A., and Pick, U. (1998) J. Biol. Chem. 273, 17553–17558). Two new proteins that are induced by iron deprivation were identified in plasma membranes of D. salina as follows: a multicopper ferroxidase termed D-Fox and an internally duplicated glycoprotein (p130B). D-Fox and p130B are accessible to glycolytic, proteolytic, and biotin surface tagging treatments, suggesting that they are surface-exposed glycoproteins. Induction of D-Fox was also manifested by ferroxidase activity in plasma membrane preparations. These results are puzzling because ferroxidases in yeast and in Chlamydomonas reinhardtii function in redox-mediated iron uptake, a mechanism that is not known to operate in D. salina. Two lines of evidence suggest that D-Fox and p130B interact with D. salina triplicated transferrin (TTf). First, chemical cross-linking combined with mass spectroscopy analysis showed that D-Fox and p130B associate with TTf and with another plasma membrane transferrin. Second, detergent-solubilized D-Fox and p130B comigrated on blue native gels with plasma membrane transferrins. 59Fe autoradiography indicated that this complex binds Fe3+ ions. Also, the induction of D-Fox and p130B is kinetically correlated with enhanced iron binding and uptake activities. These results suggest that D-Fox and p130B associate with plasma membrane transferrins forming a complex that enhances iron binding and iron uptake. We propose that the function of D-Fox in D. salina has been modified during evolution from redox-mediated to transferrin-mediated iron uptake, following a gene transfer event of transferrins from an ancestral animal cell.

Stabilization of the α2 isoform of Na,K-ATPase by mutations in a phospholipid binding pocket.

Background: The α2 isoform of Na,K-ATPase is unstable compared with α1 and α3. Results: Mutations in TM8–10 strongly stabilize α2. A novel phospholipid antagonist selectively inactivates α2, and mutations in TM8–10 protect against inactivation. Conclusion: A phosphatidylserine binding pocket within TM8–10 has been identified. Significance: Mechanistic insights into α2 instability and a possible physiological role have been obtained. The α2 isoform of Na,K-ATPase plays a crucial role in Ca2+ handling, muscle contraction, and inotropic effects of cardiac glycosides. Thus, structural, functional, and pharmacological comparisons of α1, α2, and α3 are of great interest. In Pichia pastoris membranes expressing human α1β1, α2β1, and α3β1 isoforms, or using the purified isoform proteins, α2 is most easily inactivated by heating and detergent (α2 ≫ α3 > α1). We have examined an hypothesis that instability of α2 is caused by weak interactions with phosphatidylserine, which stabilizes the protein. Three residues, unique to α2, in trans-membrane segments M8 (Ala-920), M9 (Leu-955), and M10 (Val-981) were replaced by equivalent residues in α1, singly or together. Judged by the sensitivity of the purified proteins to heat, detergent, “affinity” for phosphatidylserine, and stabilization by FXYD1, the triple mutant (A920V/L955F/V981P, called α2VFP) has stability properties close to α1, although single mutants have only modest or insignificant effects. Functional differences between α1 and α2 are unaffected in α2VFP. A compound, 6-pentyl-2-pyrone, isolated from the marine fungus Trichoderma gamsii is a novel probe of specific phospholipid-protein interactions. 6-Pentyl-2-pyrone inactivates the isoforms in the order α2 ≫ α3 > α1, and α2VFP and FXYD1 protect the isoforms. In native rat heart sarcolemma membranes, which contain α1, α2, and α3 isoforms, a component attributable to α2 is the least stable. The data provide clear evidence for a specific phosphatidylserine binding pocket between M8, M9, and M10 and confirm that the instability of α2 is due to suboptimal interactions with phosphatidylserine. In physiological conditions, the instability of α2 may be important for its cellular regulatory functions.

Plasma membrane electron transport coupled to Na(+) extrusion in the halotolerant alga Dunaliella.

The halotolerant alga Dunaliella adapts to exceptionally high salinity and maintains low [Na(+)](in) at hypersaline solutions, suggesting that it possesses efficient mechanisms for regulating intracellular Na(+). In this work we examined the possibility that Na(+) export in Dunaliella is linked to a plasma membrane electron transport (redox) system. Na(+) extrusion was induced in Dunaliella cells by elevation of intracellular Na(+) with Na(+)-specific ionophores. Elevation of intracellular Na(+) was found to enhance the reduction of an extracellular electron acceptor ferricyanide (FeCN). The quinone analogs NQNO and dicumarol inhibited FeCN reduction and led to accumulation of Na(+) by inhibition of Na(+) extrusion. These inhibitors also diminished the plasma membrane potential in Dunaliella. Anaerobic conditions elevated, whereas FeCN partially decreased intracellular Na(+) content. Cellular NAD(P)H level decreased upon enhancement of plasma membrane electron transport. These results are consistent with the operation of an electrogenic NAD(P)H-driven redox system coupled to Na(+) extrusion in Dunaliella plasma membrane. We propose that redox-driven Na(+) extrusion and recycling in Dunaliella evolved as means of adaptation to hypersaline environments.