Abstract
The halotolerant alga Dunaliella salina is unique among plants in that it utilizes a transferrin (TTf) to mediate iron acquisition (Fisher, M., Zamir, A., and Pick, U. (1998) J. Biol. Chem. 273, 17553-17558). Two new proteins that are induced by iron deprivation were identified in plasma membranes of D. salina as follows: a multicopper ferroxidase termed D-Fox and an internally duplicated glycoprotein (p130B). D-Fox and p130B are accessible to glycolytic, proteolytic, and biotin surface tagging treatments, suggesting that they are surface-exposed glycoproteins. Induction of D-Fox was also manifested by ferroxidase activity in plasma membrane preparations. These results are puzzling because ferroxidases in yeast and in Chlamydomonas reinhardtii function in redox-mediated iron uptake, a mechanism that is not known to operate in D. salina. Two lines of evidence suggest that D-Fox and p130B interact with D. salina triplicated transferrin (TTf). First, chemical cross-linking combined with mass spectroscopy analysis showed that D-Fox and p130B associate with TTf and with another plasma membrane transferrin. Second, detergent-solubilized D-Fox and p130B comigrated on blue native gels with plasma membrane transferrins. 59Fe autoradiography indicated that this complex binds Fe3+ ions. Also, the induction of D-Fox and p130B is kinetically correlated with enhanced iron binding and uptake activities. These results suggest that D-Fox and p130B associate with plasma membrane transferrins forming a complex that enhances iron binding and iron uptake. We propose that the function of D-Fox in D. salina has been modified during evolution from redox-mediated to transferrin-mediated iron uptake, following a gene transfer event of transferrins from an ancestral animal cell.
Highlights
Two major strategies of high affinity iron uptake have been demonstrated in plants and in algae as follows: siderophoremediated Fe3ϩ uptake and a redox-mediated mechanism
We found that Dunaliella ferroxidase (D-Fox) is not involved in redox-mediated iron uptake, like multicopper oxidase (MCO) in yeast or in C. reinhardtii, but it is associated with transferrins in D. salina plasma membranes and functions in enhancing iron binding
Incubation with 250 M TTM strongly inhibited ferroxidase activity of both cells and plasma membranes from iron-deficient cells, but it only slightly affected the activity of control or high salt cells (Ͻ15%, not shown). These results indicate that D-Fox is a multicopper ferroxidase and that it may be involved in iron uptake in irondeficient D. salina cells
Summary
Algal Strain and Growth Conditions—D. salina was obtained from Dr W. The reaction was terminated with 50 mM unbuffered Tris and incubated for another 10 min on ice. Protein Analysis by Two-dimensional Gel Electrophoresis— Separation and analysis of proteins on 7.5% SDS-PAGE was performed as described previously [16]. Plasma membrane protein samples (350 g of protein) were incubated in standard IEF buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 0.5% Triton X-100, 5% glycerol, 0.5% IPG buffer (GE Healthcare), and 20 mM DTT for 1 h at room temperature, loaded onto an 18-cm IPG dry strip, pH 3.0 –5.6 (GE Healthcare), by active rehydration (for 12 h), and subjected to IEF for 65,000 V-h using IPGphor instrument (GE Healthcare). Two-dimensional BN/SDS-PAGE—Membrane samples (75 g of protein) were solubilized in 30 l of buffer containing 1% Triton X-100, 50 mM Bis-tris, pH 7.0, 0.5 M ⑀-aminocaproic acid, 2 mM EDTA, 10% glycerol, and plant protease inhibitor mixture. The cells were washed twice in the same solutions and counted in a -counter
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