Abstract
High affinity iron transport in yeast is mediated by two proteins, Fet3p and Ftr1p. The multicopper oxidase Fet3p is thought to convert extracellular ferrous iron to ferric iron, which then crosses the plasma membrane through the permease Ftr1p. Fet3p is capable of oxidizing other substrates, such as p-phenylenediamine, and there is still a question of whether it is the ferroxidase activity that is essential for iron transport. Fet3p is also required for Ftr1p localization to the cell surface, making it difficult to prove a direct role for Fet3p oxidase in high affinity iron transport. In an attempt to generate Fet3p specifically lacking ferroxidase activity, we used site-directed mutagenesis to alter residues within Fet3p that had been suggested to impart iron oxidase activity. These substitutions resulted in either a loss or retention of both p-phenylenediamine and ferroxidase activities, indicating that the ability of Fet3p to act as a ferroxidase involves other amino acids. Inactive Fet3p, however, did mediate Ftr1p localization to the cell surface but did not mediate high affinity iron transport. These observations indicate that the ferroxidase activity of Fet3p is intrinsically required for high affinity iron transport.
Highlights
The high affinity iron transport system of Saccharomyces cerevisiae is specific for iron and is transcriptionally regulated by iron need (1)
Leucine 494 was changed to methionine (L494M), as found in ascorbate oxidase, or a phenylalanine (L494F), as found in many fungal laccases
All cells expressing the P4 mutant proteins showed detectable cell surface Ftr1p immunofluorescence (Fig. 4, D–J). These results indicate that all the mutant Fet3p proteins were capable of mediating the localization of Ftr1p to the cell surface
Summary
The high affinity iron transport system of Saccharomyces cerevisiae is specific for iron and is transcriptionally regulated by iron need (1). We set out to use site-directed mutagenesis to alter the substrate specificity of Fet3p in order to generate a Fet3p possessing p-phenylenediamine (pPD) oxidase activity but lacking iron oxidase activity. We reasoned that such a protein would help to determine the importance of Fet3p ferroxidase activity. Consistent with this theory, Fet3p, the only other multicopper oxidase reported to oxidize iron, possesses a leucine in the P4 position (2) To test this theory, we used site-specific mutagenesis to alter the P4 position in Fet3p. We did generate mutated Fet3p, devoid of catalytic activity that was used to further define the role of Fet3p activity in high affinity iron transport
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
