Abstract
The halotolerant alga Dunaliella salina is a recognized model photosynthetic organism for studying plant adaptation to high salinity. The adaptation mechanisms involve major changes in the proteome composition associated with energy metabolism and carbon and iron acquisition. To clarify the molecular basis for the remarkable resistance to high salt, we performed a comprehensive proteomics analysis of the plasma membrane. Plasma membrane proteins were recognized by tagging intact cells with a membrane-impermeable biotin derivative. Proteins were resolved by two-dimensional blue native/SDS-PAGE and identified by nano-LC-MS/MS. Of 55 identified proteins, about 60% were integral membrane or membrane-associated proteins. We identified novel surface coat proteins, lipid-metabolizing enzymes, a new family of membrane proteins of unknown function, ion transporters, small GTP-binding proteins, and heat shock proteins. The abundance of 20 protein spots increased and that of two protein spots decreased under high salt. The major salt-regulated proteins were implicated in protein and membrane structure stabilization and within signal transduction pathways. The migration profiles of native protein complexes on blue native gels revealed oligomerization or co-migration of major surface-exposed proteins, which may indicate mechanisms of stabilization at high salinity.
Highlights
The halotolerant alga Dunaliella salina is a recognized model photosynthetic organism for studying plant adaptation to high salinity
The halotolerant alga Dunaliella salina uses a unique osmoregulatory mechanism and is able to proliferate in environments with extreme salt content [1]
To identify changes in the membrane protein composition and organization at high salinity, we compared membranes obtained from cells cultured either at 0.5 M or at 3.0 M NaCl
Summary
D. salina, a green species, was obtained from the culture collection of Dr W. The cells were osmotically lysed (1:4 dilution) by suspension of the pellet at a final concentration of 21⁄7108 cells/ml in bursting buffer (10 mM Naϩ-MOPS, pH 7.2, 10 mM KCl, 2 mM MgCl2, 5 mM -aminocaproic acid, 1 mM benzamidine, and plant protease inhibitor mixture (Sigma, catalog number P-9599) diluted to 1:200. The pellet, which contained plasma membranes, was washed by suspension buffer (0.5 M glycerol, 10 mM Naϩ-MOPS, pH 7.2, 2 mM MgCl2, and 10 mM KCl) and centrifuged again as above. Hits produced by matching of at least one peptide with a minimum score of 40 were considered borderline and subjected to subsequent validation by de novo sequencing and MS BLAST database searching as described previously [23]. When proteins were solely identified with MS BLAST, at least one high scoring segment pair with the score above 72 and the total score of the hit above 200 were required
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