Abstract
Heat shock protein 90 (Hsp90) is a molecular chaperone highly conserved across the species from prokaryotes to eukaryotes. Hsp90 is essential for cell viability under all growth conditions and is proposed to act as a hub of the signaling network and protein homeostasis of the eukaryotic cells. By interacting with various client proteins, Hsp90 is involved in diverse physiological processes such as signal transduction, cell mobility, heat shock response and osmotic stress response. In this research, we cloned the dshsp90 gene encoding a polypeptide composed of 696 amino acids from the halotolerant unicellular green algae Dunaliella salina. Sequence alignment indicated that DsHsp90 belonged to the cytosolic Hsp90A family. Further biophysical and biochemical studies of the recombinant protein revealed that DsHsp90 possessed ATPase activity and existed as a dimer with similar percentages of secondary structures to those well-studied Hsp90As. Analysis of the nucleotide sequence of the cloned genomic DNA fragment indicated that dshsp90 contained 21 exons interrupted by 20 introns, which is much more complicated than the other plant hsp90 genes. The promoter region of dshsp90 contained putative cis-acting stress responsive elements and binding sites of transcriptional factors that respond to heat shock and salt stress. Further experimental research confirmed that dshsp90 was upregulated quickly by heat and salt shock in the D. salina cells. These findings suggested that dshsp90 might serve as a component of the early response system of the D. salina cells against environmental stresses.
Highlights
The unicellular green algae Dunaliella is the most halotolerant eukaryote known, and can survive in media of a wide range of chemical compositions and salt concentrations ranging from 0.05 M to saturation
The full length gene was obtained by 5’ and 3’ RACEs to amplify the flanking regions using the primers listed in Table S1, which resulted in a 2395 bp cDNA including an ATG start codon, a TAA stop codon, a 41 bp 5’ untranslated region (UTR) and a 263 bp 3’ UTR
A comparison of the genomic sequence with the cDNA sequence of dshsp90 revealed that the ORF of dshsp90 contained 21 exons interrupted by 20 introns with an average size of 313 bp, which is longer than the mean length of higher plant introns (249 bp) [24] but smaller than that of C. reinhardtii (373 bp)
Summary
The unicellular green algae Dunaliella is the most halotolerant eukaryote known, and can survive in media of a wide range of chemical compositions and salt concentrations ranging from 0.05 M to saturation. Compared with the other general Hsps, Hsp interact with a subset of proteins, and more than 200 substrate ( called client) proteins have been identified These client proteins are involved in diverse physiological/pathological processes such as signal transduction, cell mobility, tumorigenesis, steroid signaling, innate immunity, heat shock response and telomere maintenance [15,16,17,18]. In Saccharomyces cerevisiae, Hsp is found to be required for the high osmotic stress response and regulates a novel signaling pathway via the co-chaperone Cdc37p parallel to the HOG pathway [20,21]. Considering that Hsp70- and Hsp90-like proteins were upregulated in Dunaliella under halophilic conditions [11] and the importance of Hsp in the stress response of eukaryotic cells, it is possible that Dunaliella Hsp (DsHsp90) is involved in the halotolerance of Dunaliella. DsHsp belongs to the Hsp90A family and is involved in the adaption of Dunaliella against extreme environmental conditions
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