Specific aspartate residues in FET3 control high‐affinity iron transport in Saccharomyces cerevisiae

Abstract

Site-directed mutagenesis was performed on a set of six aspartate residues of Fet3, the multicopper ferroxidase involved in high-affinity iron transport in Saccharomyces cerevisiae, in order to comprehend the molecular determinants of the protein function. Asp312, Asp315, Asp319 and Asp320 were predicted by homology modelling to be located in a negatively charged surface-exposed loop of the protein. Other two aspartate residues (Asp278 and Asp279) are placed close to the type 1 copperand iron-binding sites, possibly linking these sites to the negatively charged region. In vivo results showed that mutation of Asp319 and Asp320 to yield D319N and D320N derivatives strongly impairs the ability of the yeast to grow under ironlimiting conditions. In particular, substitution of Asp320 with asparagine essentially abolished the Fet3-dependent iron transport activity. All other mutants (D278Q, D279N, D312N and D315I) behaved essentially as the wild-type protein. The electron paramagnetic resonance spectrum of the soluble forms of D319N and D320N showed significant changes of the copper sites' geometry in D319N but not in D320N. At variance with the membrane-bound forms, soluble D319N and D320N derivatives were highly susceptible to proteolytic degradation, suggesting that replacement of Asp319 or Asp320 locally modifies the structure of Fet3, making the protein sensitive to proteolysis when it is not protected by the membrane environment. In turn, this might be evidence of a shielding role of the permease Ftr1, which could interact with Fet3 at the level of the aspartate-rich negatively charged region.

IRON MAN is a ubiquitous family of peptides that control iron transport in plants

“…Although the exact molecular mechanism by which IMAs regulate Fe uptake genes remain to be elucidated, binding to Fe 2+ may control the stability of IMA peptides and thereby regulate Fe uptake. Negatively charged residues such as aspartate participate in the Fe coordination in numerous proteins 52,53,54 . Consistent with the putative metal-binding properties of the Asp stretch in the IMA motif, we showed that IMA peptides can bind Fe 2+ and other metals.…”

The copper-iron connection in biology: Structure of the metallo-oxidase Fet3p

“…However, all of these residues are part of the water-accessible (Connolly) surface of the protein, ensuring access from bulk solvent. A previous modeling study led to the proposal that D319 and D320 are part of a solvent-exposed, negative patch involved in Fe binding (62). In fact, these residues are part of an electrostatic ''zipper'' where the two carboxylate side chains are in salt bridges with the side chains of R463 and R427, respectively.…”

Involvement of Arabidopsis Multi-Copper Oxidase-Encoding LACCASE12 in Root-to-Shoot Iron Partitioning: A Novel Example of Copper-Iron Crosstalk

“…Based on site-directed mutagenesis of the S. cerevisae FET3 protein, previous research identified D319 and D320 to be critical for the growth of yeast under Fe-deficient conditions. In particular, mutation of D320 abrogated the FET3-dependent Fe uptake function (Bonaccorsi di Patti et al, 2005;Quintanar et al, 2007). Kinetic studies of mutations of the E185, D283, and D409 residues, respectively, showed their important role in Fe(II) oxidation (Quintanar et al, 2007).…”

“…S. cerevisiae ferroxidase FET3(Askwith et al, 1994) contains essential aspartic residues which play an important role in Fe(II) oxidation(Bonaccorsi di Patti et al, 2005;Quintanar et al, 2007).Frontiers in Plant Science | www.frontiersin.org…”