The purpose of this study was to find out whether protein kinases are involved in the osmotic response of the halotolerant green alga Dunaliella salina.Hyperosmotic shock leads to a rapid transient activation of two different protein kinases, identified by an in-gel assay: A 40 kD kinase (PK 40), which is activated within seconds after osmotic shocks, and a 74 kD kinase (PK 74), activated a few minutes after the shock. Subcellular fractionation shows that PK 40 is a soluble enzyme whereas PK 74 is a membrane-associated protein localized in a plasma membrane-enriched microsomal fraction. Heat shock and cytoplasmic acidification activate a 40 kD, but not a 74 kD protein kinase. Both PK 40 and PK 74 phosphorylate histone at threonine residues and do not require calcium ions.The following results suggest that PK 74 may be a novel type of a MEK 1 kinase: 1) PK 74 phosphorylates ERK 1, a specific substrate of mammalian MEK 1, and 2). Antibodies against a conserved peptide of MEK 1 cross-react specifically with a 74kD component localized at the plasma membrane fraction, which possesses the PK 74 activity.It is suggested that PK 74 is activated by osmotically-induced structural changes at the plasma membrane. Its possible role in the osmotic signal transduction is discussed.

The accompanying papers (Keyhani, N. O., and Roseman, S. (1996) J. Biol. Chem. 271, 33414-33424; Keyhani, N. O., and Roseman, S. (1996) J. Biol. Chem. 271, 33425-33432) describe two unique β-N-acetylglucosaminidases from Vibrio furnissii. A third, ExoII, is reported here. The gene, exoII, was cloned into Escherichia coli, sequenced, and ExoII purified to apparent homogeneity (36 kDa). The molecular weight and N-terminal 16 amino acids of the protein conform to the predicted sequence. ExoII exhibited unique substrate specificity. It rapidly cleaved p-nitrophenyl and 4-methylumbelliferyl β-GlcNAc, was slightly active with p-nitrophenyl-β-GalNAc, and was inactive with all other GlcNAc derivatives tested, including N,N′-diacetylchitobiose and (GlcNAc)n, n = 3-6. Unlike GlcNAc (Ki, 210 μM), (GlcNAc)n are poor inhibitors of ExoII. The predicted protein sequence is unique among β-N-acetylglucosaminidases excepting Cht60, recently cloned from a marine Alteromonas (Tsujibo, H., Fujimoto, K., Tanno, H., Miyamoto, K., Imada, C., Okami, Y., and Inamori, Y. (1994) Gene (Amst.) 146, 111-115). Cht60, a chitobiase, is 26.9% identical to ExoII in a 182-amino acid overlap, but the two enzymes differ in substrate specificity and other properties. ExoII shares similarity with five bacterial and yeast β-glucosidases, up to 44% identity in the 25-amino acid catalytic domain. By analogy, ExoII may play a role in signal transduction between invertebrate hosts and V. furnissii.

Changes in phosphometabolites, following osmotic shock, were analyzed by two-dimensional thin layer chromatography, in extracts of the halotolerant alga Dunaliella salina in order to clarify the regulation of glycerol synthesis from starch. The experiments were carried out in wild-type and in osmotically defective mutant cells. It is demonstrated that hyperosmotic shock induces a decrease in fructose 6-phosphate and an increase in fructose-1,6-bisphosphate indicating the activation of phosphofructokinase. Two mutants, which are specifically defective in their response to hyperosmotic shock, accumulate glucose 6-phosphate or phosphogluconate following shock, and have remarkably reduced activities of glucose-6-phosphate dehydrogenase and of phosphogluconate dehydrogenase, respectively. These results indicate that the pentose-phosphate oxidative pathway has a major role in glycerol synthesis. Hyperosmotic shock leads to a transient accumulation of phosphorylcholine and to a decrease of inositolbisphosphate in D. salina extracts. Accumulation of phosphorylcholine is not detected in osmotically defective mutants. Hypoosmotic shock induces an increase in inositolbisphosphate but not in phosphorylcholine. These results are consistent with previous indications for differential activations of phospholipases by hyper or hypoosmotic shock in Dunaliella. Based on these results we suggest that (a) phosphofructokinase is an important checkpoint enzyme in the regulation of glycerol production, and (b) that the pentose-phosphate pathway has a major role in keeping oxidation-reduction balance during glycerol synthesis. The possible role of lipid breakdown products as second messengers in regulating glycerol production in Dunaliella is discussed.

Different microorganisms, including yeast and algae, accumulate large amounts of polyphosphates. However, the physiological role of polyphosphates is largely unknown. In vivo 31P NMR studies, carried out in the unicellular alga, Dunaliella salina, demonstrate that cytoplasmic alkalization induces massive hydrolysis of polyphosphates, which is correlated kinetically with the recovery of cytoplasmic pH. Analysis of acid extracts of the cells indicates that long-chain polyphosphates are hydrolysed mainly to tripolyphosphate. It is suggested that the hydrolysis of polyphosphates provides a pH-stat mechanism to counterbalance alkaline stress.

A technique for selection of Dunaliella mutants defective in their capacity to recover from osmotic shocks has been developed. The selection is based on physical separation of mutants on density gradients. This technique takes advantage of the fact that Dunaliella cells, when exposed to osmotic shocks, initially change volume and density due to water gain or loss and subsequently recover their volume and density by readjusting their intracellular glycerol. Eight mutants that do not recover their original density following hyperosmotic shocks have been isolated. The mutants grow similar to wild type cells in 1 molar NaCl, and recover like the wild type from hypotonic shocks but are defective in recovering from hypertonic shocks. A partial characterization of one of the mutants is described.