Salt induction of fatty acid elongase and membrane lipid modifications in the extreme halotolerant alga Dunaliella salina

Abstract

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.

Figure 1

Sequence alignment of Kcs from D. salina and plant Kcs/Fae proteins. Protein sequences were aligned by the use of the PILEUP program (Genetics Computer Group, Madison, WI). GenBank accession numbers for the aligned proteins are: Fae from Brassica napus, U50771; Fae1 from Arabidopsis, U29142; Kcs from Simmondsia chinesis, U37088; Cut1 from Arabidopsis, AF129511 (recently, CUT1 was shown to be identical to CER6, a gene critical for Arabidopsis pollination; Fiebig et al., 2000); Kcs1 from Arabidopsis, AF053345; FDH from Arabidopsis, ATH010713; and Kcs2 from D. salina (denoted elsewhere in this paper as Kcs), AF333040. Black circles, Conserved Cys; white circles, conserved Cys with one exception.

Figure 2

Acyl-CoA dependence of Kcs activity. A, Dependence on Acyl-CoA concentration. Assay mixtures, with 30 μg of protein of fraction 1 from cells grown in 3.5 m NaCl, contained the indicated concentrations of acyl(18:1)-CoA. Assay conditions and product analysis were as described in “Materials and Methods.” B, Kcs activity with different acyl-CoAs. Activity assays, as described in A, were performed with 100 μm of the acyl-CoAs indicated. sd values are indicated for acyl(18:1)-CoA and acyl(16:0)-CoA substrates. Kcs activity, cpm in 0.6 mL of chloroform extracts, as detailed in “Materials and Methods.”

Figure 3

Analysis of kcs transcript and Kcs activity in D. salina cells after exposure to high salt. Cultures were transferred, in two steps, from a medium with 0.5 m to a medium with 3.5 m NaCl. Top, Assay of Kcs activity. Cell culture batches of approximately 1 L were removed at the indicated times after transfer to 3.5 m NaCl and the cells were fractionated to give fraction 1. Kcs activity was determined with 30 μg of protein of fraction 1 and 100 μm acyl(16:0)-CoA (white bars) or acyl(18:1)-CoA (black bars) as described in “Materials and Methods.” Bottom, Northern-blot analysis of kcs mRNA. Samples of approximately 200 mL were removed at the indicated times after transfer to 3.5 m NaCl and total RNA was extracted. Northern-blot hybridization with kcs cDNA (kcs) or standard (std) probe was as described in “Materials and Methods.”

Figure 4

Analysis of Kcs reaction products. Kcs assay mixtures with 100 μm of the indicated Acyl-CoAs were supplemented with 0.5 mm each NADH and NADPH. Assay conditions, reaction product processing, resolution of the reaction products by thin-layer chromatography (TLC), and autoradiography were as described in “Materials and Methods,” Markers: 16:0, methyl ester of [³H] palmitic acid; 18:0, methyl ester of [¹⁴C] stearic acid.

Figure 5

Expression and activity of Kcs in E. coli. A, Detection of heterologously expressed Kcs. The kcs cDNA cloned in the pET28c expression vector was transformed into E. coli. Transformants with the recombinant plasmid (+) or vector alone (−) were metabolically labeled with [³⁵S]Met and membrane fractions were analyzed by SDS-PAGE and autoradiography as described in “Materials and Methods.” The major translation product is marked by an arrow. A preferentially labeled >107-kD polypeptide is likely to represent an incompletely dissociated dimeric form of Kcs, as discussed in “Results.” B, E. Coli transformants with the kcs cDNA containing plasmid (black symbols), or the pET28c vector alone (white symbols), were treated under conditions inducing kcs expression and a membrane fraction was isolated and assayed for Kcs activity as described in “Materials and Methods.” Reaction mixtures contained 3 (rectangles) or 6 (circles) μg of protein of the E. coli fraction.