The halotolerant alga Dunaliella salina can adapt to practically the entire range of salt concentrations. This capacity is achieved primarily by massive accumulation of glycerol and by efficient elimination of sodium ions, which require major metabolic investment. To clarify the molecular mechanisms of salinity tolerance in D. salina, we performed a proteomic differential analysis aimed to identify salt-regulated proteins in different sub-cellular fractions. Soluble proteins were identified by 2D IEF/SDS-PAGE combined with MALDI-TOF MS/MS, whereas plasma membrane proteins were biotin-tagged, separated by blue-native/SDS-PAGE, and identified by nano-LC/MS-MS. High salt up-regulated plasma membrane carbonic anhydrases, which mediate bicarbonate acquisition, key enzymes in Calvin cycle, starch mobilization and redox energy production. These results indicated that D. salina enhances photosynthetic CO2 assimilation and diverts its principle carbon and energy resources for synthesis of glycerol. In the plasma membrane we identified: (i) bacterial-type surface coat proteins (peptidoglycan-associated lipoprotein) and tubulin, which probably function in stabilizing the membrane against osmotic lysis; (ii) small GTP-binding proteins which may be involved in signal-transduction in response to salt/osmotic stress; (iii) lipid metabolizing enzymes, possibly associated with osmotic sensing; (iv) chaperones and proteolytic enzymes probably involved in enhancing the turnover and stabilization of membrane proteins at high salinity; (v) ion transporters for protons, iron, nitrate, ammonium and possibly sodium. 342Taken together, these results suggest that concerted changes in multiple pathways contribute to unique ability of D. salina to withstand high salinity. A surprising observation was the finding that high salinity induced iron deficiency stress in D. salina. We discovered that D. salina evolved special strategies to cope with iron limitation. It utilizes a unique mechanism for iron acquisition, via membrane-associated transferrins, that bind and internalize ferric ions into acidic vacuoles. In the chloroplast, iron deprivation induced one major protein, identified as a PS-I chlorophyll a/b-binding protein, which largely increased the size of PS-I units. Interestingly, iron-deprived cyanobacteria, accumulate in PS-I a different type of chlorophyll-binding protein. This may represent a general strategy of photosynthetic organisms to adapt to iron deprivation.

Uptake of iron in the halotolerant alga Dunaliella salina is mediated by a transferrin-like protein (TTf), which binds and internalizes Fe31 ions. Recently, we found that iron deficiency induces a large enhancement of iron binding, which is associated with accumulation of three other plasma membrane proteins that associate with TTf. In this study, we characterized the kinetic properties of iron binding and internalization and identified the site of iron internalization. Iron deficiency induces a 4-fold increase in Fe binding, but only 50% enhancement in the rate of iron uptake and also increases the affinity for iron and bicarbonate, a coligand for iron binding. These results indicate that iron deprivation leads to accumulation and modification of iron-binding sites. Iron uptake in iron-sufficient cells is preceded by an apparent time lag, resulting from prebound iron, which can be eliminated by unloading iron-binding sites. Iron is tightly bound to surface-exposed sites and hardly exchanges with medium iron. All bound iron is subsequently internalized. Accumulation of iron inhibits further iron binding and internalization. The vacuolar inhibitor bafilomycin inhibits iron uptake and internalization. Internalized iron was localized by electron microscopy within vacuolar structures that were identified as acidic vacuoles. Iron internalization is accompanied by endocytosis of surface proteins into these acidic vacuoles. A novel kinetic mechanism for iron uptake is proposed, which includes two pools of bound/compartmentalized iron separated by a rate-limiting internalization stage. The major parameter that is modulated by iron deficiency is the iron-binding capacity. We propose that excessive iron binding in iron-deficient cells serves as a temporary reservoir for iron that is subsequently internalized. This mechanism is particularly suitable for organisms that are exposed to large fluctuations in iron availability. Iron is an essential element for survival for all living organisms, including photosynthetic organisms that have a special requirement for iron as a cofactor of multiple elements in their electron transport system. Because of its low solubility in aerobic solutions, iron is recognized as a major limiting factor for prolifera- tion of plants and algae. To counterbalance iron lim- itation, photosynthetic organisms evolved efficient high-affinity iron uptake mechanisms that are induced under iron limitation. Two major mechanisms of iron acquisition studied in plants are based either on re-

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.

Uptake of iron in the halotolerant alga Dunaliella salina is mediated by a transferrin-like protein (TTf), which binds and internalizes Fe(3+) ions. Recently, we found that iron deficiency induces a large enhancement of iron binding, which is associated with accumulation of three other plasma membrane proteins that associate with TTf. In this study, we characterized the kinetic properties of iron binding and internalization and identified the site of iron internalization. Iron deficiency induces a 4-fold increase in Fe binding, but only 50% enhancement in the rate of iron uptake and also increases the affinity for iron and bicarbonate, a coligand for iron binding. These results indicate that iron deprivation leads to accumulation and modification of iron-binding sites. Iron uptake in iron-sufficient cells is preceded by an apparent time lag, resulting from prebound iron, which can be eliminated by unloading iron-binding sites. Iron is tightly bound to surface-exposed sites and hardly exchanges with medium iron. All bound iron is subsequently internalized. Accumulation of iron inhibits further iron binding and internalization. The vacuolar inhibitor bafilomycin inhibits iron uptake and internalization. Internalized iron was localized by electron microscopy within vacuolar structures that were identified as acidic vacuoles. Iron internalization is accompanied by endocytosis of surface proteins into these acidic vacuoles. A novel kinetic mechanism for iron uptake is proposed, which includes two pools of bound/compartmentalized iron separated by a rate-limiting internalization stage. The major parameter that is modulated by iron deficiency is the iron-binding capacity. We propose that excessive iron binding in iron-deficient cells serves as a temporary reservoir for iron that is subsequently internalized. This mechanism is particularly suitable for organisms that are exposed to large fluctuations in iron availability.