Origin of ß-carotene-rich plastoglobuli in Dunaliella bardawil.

The halotolerant microalgae Dunaliella bardawil accumulates under nitrogen deprivation two types of lipid droplets: plastoglobuli rich in ß-carotene (ßC-plastoglobuli) and cytoplasmatic lipid droplets (CLDs). We describe the isolation, composition, and origin of these lipid droplets. Plastoglobuli contain ß-carotene, phytoene, and galactolipids missing in CLDs. The two preparations contain different lipid-associated proteins: major lipid droplet protein in CLD and the Prorich carotene globule protein in ßC-plastoglobuli. The compositions of triglyceride (TAG) molecular species, total fatty acids, and sn-1+3 and sn-2 positions in the two lipid pools are similar, except for a small increase in palmitic acid in plastoglobuli, suggesting a common origin. The formation of CLD TAG precedes that of ßC-plastoglobuli, reaching a maximum after 48 h of nitrogen deprivation and then decreasing. Palmitic acid incorporation kinetics indicated that, at early stages of nitrogen deprivation, CLD TAG is synthesized mostly from newly formed fatty acids, whereas in ßC-plastoglobuli, a large part of TAG is produced from fatty acids of preformed membrane lipids. Electron microscopic analyses revealed that CLDs adhere to chloroplast envelope membranes concomitant with appearance of small ßC-plastoglobuli within the chloroplast. Based on these results, we propose that CLDs in D. bardawil are produced in the endoplasmatic reticulum, whereas ßC-plastoglobuli are made, in part, from hydrolysis of chloroplast membrane lipids and in part, by a continual transfer of TAG or fatty acids derived from CLD.

A transferrin-like protein that does not bind iron is induced by iron deficiency in the alga Dunaliella salina.

Iron deficiency induces two major transferrin-like proteins in the plasma membrane (Pm) of the halotolerant alga Dunaliella salina. TTf, a 150-kDa protein, previously identified as a salt-induced triplicated transferrin, having iron-binding characteristics resembling animal transferrins, and a 100-kDa protein designated idi-100 (for iron-deficiency-induced 100 kDa protein). According to the predicted amino acid sequence of idi-100, it is only 30% identical to TTf and differs from it in having two, rather than three, homologous internal repeats and in a lower conservation of canonical iron/bicarbonate binding residues. Both are localized in the outer surface of the membrane; however, TTf can be dissociated from the membrane by treatment with EDTA, whereas release of idi-100 requires detergents. The accumulation of idi-100 under iron deficiency lags behind that of TTf and in contrast to TTf, it is not induced by high salinity, suggesting that induction of idi-100 requires lower Fe threshold levels than that of TTf. In contrast to TTf, idi-100 does not bind Fe; however, there are indications for interactions with bicarbonate ions. These results suggest that despite their common resemblance to transferrins, their similar subcellular localization and their induction by iron deficiency, idi-100 and TTf fulfill different functions.

Natural protein engineering: a uniquely salt-tolerant, but not halophilic, alpha-type carbonic anhydrase from algae proliferating in low- to hyper-saline environments.

Dunaliella salina is a unicellular green alga thriving in environments ranging from fresh water to hyper-saline lakes, such as the Dead Sea. An unusual, internally duplicated, 60 kDa alpha-type carbonic anhydrase (dCA I), located on the surface of this alga, is expected to function over a broad range of salinities. It would therefore differ from other carbonic anhydrases that already lose activity at low salinities and also from halophilic proteins that require high salinities for conformational stability. Enzymatic analyses indeed indicated that dCA I retained activity at salt concentrations ranging from low salt to at least 1.5 M NaCl or KCl for CO(2) hydration, 2.0 M NaCl for esterase activity and 0.5 M for bicarbonate dehydration. Although measurements at higher salinities were constrained by the interference of salt in the respective assayed reactions, activity was noticeable even at 4.0 M NaCl. Comparisons of the internally duplicated dCA I to single-domain derivatives indicated that inter-domain interactions played a decisive role in the stability, activity, salt tolerance and pH responses of dCA I. Hence dCA I is a uniquely salt- tolerant protein, retaining an active conformation over a large range of salinities and, as a Zn metalloenzyme, largely immune to the specific inhibitory effects of anions. Its unique features make dCA I a useful model to understand the physico-chemical basis of halotolerance and protein-salt interactions in general.

Three-dimensional structure of a halotolerant algal carbonic anhydrase predicts halotolerance of a mammalian homolog.

Protein molecular adaptation to drastically shifting salinities was studied in dCA II, an alpha-type carbonic anhydrase (EC 4.2.1.1) from the exceptionally salt-tolerant unicellular green alga Dunaliella salina. The salt-inducible, extracellular dCA II is highly salt-tolerant and thus differs from its mesophilic homologs. The crystal structure of dCA II, determined at 1.86-A resolution, is globally similar to other alpha-type carbonic anhydrases except for two extended alpha-helices and an added Na-binding loop. Its unusual electrostatic properties include a uniformly negative surface electrostatic potential of lower magnitude than that observed in the highly acidic halophilic proteins and an exceptionally low positive potential at a site adjoining the catalytic Zn(2+) compared with mesophilic homologs. The halotolerant dCA II also differs from typical halophilic proteins in retaining conformational stability and solubility in low to high salt concentrations. The crucial role of electrostatic features in dCA II halotolerance is strongly supported by the ability to predict the unanticipated halotolerance of the murine CA XIV isozyme, which was confirmed biochemically. A proposal for the functional significance of the halotolerance of CA XIV in the kidney is presented.

An unusual halotolerant alpha-type carbonic anhydrase from the alga Dunaliella salina functionally expressed in Escherichia coli.

A 60-kDa, salt-inducible, internally duplicated alpha-type carbonic anhydrase (Dca) is associated with the plasma membrane of the extremely salt-tolerant, unicellular, green alga Dunaliella salina. Unlike other carbonic anhydrases, Dca remains active over a very broad range of salinities (0-4M NaCl), thus representing a novel type of extremely halotolerant enzyme. To elucidate the structural principles of halotolerance, structure-function investigations of Dca have been initiated. Such studies require considerable amounts of the enzyme, and hence, large-scale algal cultivation. Furthermore, the purified enzyme is often contaminated with other, co-purifying algal carbonic anhydrases. Expression in heterologous systems offers a means to produce, and subsequently purify, sufficiently large amounts of Dca required for activity and structural studies. Attempts to over-express Dca in the Escherichia coli BL21(DE3)pLysS strain, after optimizing various expression parameters, produced soluble, but weakly active protein, composed of fully reduced and variably -S-S- cross-linked chains (each of the Dca repeats contains a pair of cysteine residues, presumably forming a disulfide bond). However, when the E. coli Origami B(DE3)pLysS strain was used as a host, a functionally active enzyme with proper disulfide bonds was formed in good yield. Affinity-purified recombinant Dca resembled the native enzyme from D. salina in activity and salt tolerance. Hence, this expression system offers a means of pursuing detailed studies of this extraordinary protein using biochemical, biophysical, and crystallographic approaches.

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.

Identification, cDNA cloning, expression, crystallization and preliminary X-ray analysis of an exceptionally halotolerant carbonic anhydrase from Dunaliella salina.

An extracellular alpha-type carbonic anhydrase (dCAII) from the salt-tolerant alga Dunaliella salina differs from its mesophilic counterparts in remaining active from zero to multimolar salt concentrations. To gain insight into the outstanding salt tolerance of dCAII, the enzyme was functionally overexpressed in Escherichia coli, purified by affinity chromatography and crystallized by the hanging-drop method. The crystals belonged to space group P2(1), with unit-cell parameters a = 47.0, b = 119.9, c = 58.5 A, beta = 94.2 degrees. Data from a single crystal were collected to 2.4 A resolution under cryogenic conditions (120 K) using an R-AXIS IV(++) detector mounted on a Rigaku RU-H3R rotating-anode generator. The asymmetric unit contains two molecules of the protein, which corresponds to V(M) = 2.65 A(3) Da(-1) and a solvent content of 52.7%.