Abstract
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.
Highlights
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
In the green algae Chlorella and Chlamydomonas and, in several diatoms, iron deficiency induces a large enhancement of ferrireductase activity associated with stimulation of highaffinity iron uptake, suggesting a redox-driven iron uptake mechanism similar to strategy I plants (Allnutt and Bonner, 1987; Eckhardt and Buckout, 1998; Lynnes et al, 1998)
Comprehensive identification of proteins and genes associated with iron acquisition was made only in two species of algae, Chlamydomonas reinhardtii and Dunaliella salina
Summary
The time course of iron uptake in D. salina reveals an initial time lag of 15 to 20 min This time lag was apparent in control cells, which have been cultured with iron, but was absent in iron-deficient cells (Fig. 1A). We found that preincubation of control D. salina cells for 30 to 60 min in iron-deficient medium almost completely eliminated this apparent time lag (Fig. 1B). Preincubation of iron-deficient cells with iron introduced a similar time lag in the onset of iron uptake (Fig. 1C). Preincubated for 1 h in the light in Fe-deficient medium, for unloading occupied iron-binding sites (PI) or for 20 min on ice with saturating Fe citrate to upload unoccupied iron-binding sites followed by washing (Fe-Cit.) After these treatments, cells were incubated with 59Fe31 citrate at room temperature for the indicated times and analyzed for internalized iron content.
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