title: 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 copper- and 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 iron-limiting 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.
authors: Department of Biochemical Sciences, University of Rome La Sapienza, p.le A. Moro 5-00185, Rome, Italy.
authors: Maria Carmela
authors: MC
authors: Bonaccorsi di Patti
authors: Maria Rosa
authors: MR
authors: Felice
authors: Ivana
authors: I
authors: De Domenico
authors: Amalia
authors: A
authors: Lania
authors: Flaminia
authors: F
authors: Alaleona
authors: Giovanni
authors: G
authors: Musci
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002570:Ceruloplasmin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D017136:Ion Transport; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014170:Transformation, Genetic
chemical_list: C098723:FTR1 protein, S cerevisiae; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D007501:Iron; D002570:Ceruloplasmin; C085513:FET3 protein, S cerevisiae