Irena Gokhman

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.

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 β-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 thekcs mRNA. The function of the kcsgene product was directly demonstrated by the condensing activity exhibited by Escherichia coli cells expressing thekcs cDNA. The effect of salinity on kcsexpression 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 mNaCl contained a considerably higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids compared with cells grown in 0.5m 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.

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.

Cis interaction between Semaphorin6A and Plexin‐A4 modulates the repulsive response to Sema6A

The correct navigation of axons to their targets depends on guidance molecules in the extra‐cellular environment. Differential responsiveness to a particular guidance cue is largely an outcome of disparity in the expression of its receptors on the reacting axons. Here, we show that the differential responsiveness of sympathetic and sensory neurons to the transmembrane Semaphorin Sema6A is mainly determined by its co‐expression in the responding neurons. Both sympathetic and sensory neurons express the Sema6A receptor Plexin‐A4, but only sympathetic neurons respond to it. The expression of Sema6A counteracts this responsiveness and is detected only in sensory neurons. Remarkably, sensory neurons that lack Sema6A gain sensitivity to it in a Plexin‐A4‐dependent manner. Using heterologus systems, we show that the co‐expression of Sema6A and Plexin‐A4 hinders the binding of exogenous ligand, suggesting that a Sema6A–Plexin‐A4 cis interaction serves as an inhibitory mechanism. Finally, we provide evidence for differential modes of interaction in cis versus in trans. Thus, co‐expression of a transmembrane cue together with its receptor can serve as a guidance response modulator.

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

A 60-kDa, salt-inducible, internally duplicated a-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 (0–4 M 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.

ADAM metalloproteases promote a developmental switch in responsiveness to the axonal repellant Sema3A

During embryonic development, axons can gain and lose sensitivity to guidance cues, and this flexibility is essential for the correct wiring of the nervous system. Yet, the underlying molecular mechanisms are largely unknown. Here we show that receptor cleavage by ADAM (A Disintegrin And Metalloprotease) metalloproteases promotes murine sensory axons loss of responsiveness to the chemorepellant Sema3A. Genetic ablation of ADAM10 and ADAM17 disrupts the developmental downregulation of Neuropilin-1 (Nrp1), the receptor for Sema3A, in sensory axons. Moreover, this is correlated with gain of repulsive response to Sema3A. Overexpression of Nrp1 in neurons reverses axonal desensitization to Sema3A, but this is hampered in a mutant Nrp1 with high susceptibility to cleavage. Lastly, we detect guidance errors of proprioceptive axons in ADAM knockouts that are consistent with enhanced response to Sema3A. Our results provide the first evidence for involvement of ADAMs in regulating developmental switch in responsiveness to axonal guidance cues.

Distinct Cytoplasmic Domains in Plexin-A4 Mediate Diverse Responses to Semaphorin 3A in Developing Mammalian Neurons

Deletion analysis reveals how one neuronal guidance cue–receptor pair can produce either axonal retraction or dendritic branching. Receptor Domains for Neuronal Retraction or Branching Various extracellular cues guide neuronal migration and circuit formation. Semaphorin 3A signaling through its receptor Plexin-A4 can elicit either growth cone collapse in the axons of developing sensory neurons or growth and branching in the dendrites of cortical neurons. By expressing various Plexin-A4 mutant proteins in neuronal cultures from Plexin-A4–deficient mice, Mlechkovich et al. identified specific cytoplasmic domains of the receptor responsible for these distinct responses. Although the guanine nucleotide exchange factor FARP2 was involved in both responses, axonal collapse was less dependent on the intracellular FARP2-binding motif in Plexin-A4 than was dendritic growth and branching. Additionally, axon growth cone collapse required a different combination of Plexin-A4 cytoplasmic domains than were required for dendritic arborization. Although the downstream mechanism requires further investigation, the findings show how the composition of receptor cytoplasmic domains can generate different cellular responses to the same ligand. Thus, multiple parallel, intracellular signaling pathways may mediate semaphorin 3A–induced functions, but the responses are first diversified by the Plexin-A4 receptor. Guidance receptor signaling is crucial for neural circuit formation and elicits diverse cellular events in specific neurons. We found that signaling from the guidance cue semaphorin 3A diverged through distinct cytoplasmic domains in its receptor Plexin-A4 to promote disparate cellular behavior in different neuronal cell types. Plexin-A4 has three main cytoplasmic domains—C1, Hinge/RBD, and C2—and interacts with family members of the Rho guanine nucleotide exchange factor FARP proteins. We show that growth cone collapse occurred in Plexin-A4–deficient dorsal root ganglion sensory neurons reconstituted with Plexin-A4 containing either the Hinge/RBD or C2 domain, whereas both of the Hinge/RBD and C1 domains were required for dendritic arborization in cortical neurons. Although knockdown studies indicated that both the collapse and arborization responses involved FARP2, mutations in the cytoplasmic region of Plexin-A4 that reduced its interaction with FARP2 strongly inhibited semaphorin 3A–induced dendritic branching but not growth cone collapse, suggesting that different degrees of interaction are required for the two responses or that developing axons have an indirect path to FARP2 activation. Thus, our study provided insights into the multifunctionality of guidance receptors, in particular showing that the semaphorin 3A signal diverges through specific functions of the modular domains of Plexin-A4.

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%.