Structural requirements for the activity of the MirB ferrisiderophore transporter of Aspergillus fumigatus.
Eukaryot Cell
; 11(11): 1333-44, 2012 Nov.
Article
en En
| MEDLINE
| ID: mdl-22903978
ABSTRACT
Siderophores have been identified as virulence factors in the opportunistic fungal pathogen Aspergillus fumigatus. The 14-pass transmembrane protein MirB is postulated to function as a siderophore transporter, responsible for uptake of the hydroxamate siderophore N,N',Nâ³-triacetylfusarinine C (TAFC). Our aim was to identify amino acids of A. fumigatus MirB that are crucial for uptake of TAFC. Site-directed mutagenesis was used to create MirB mutants. Expression of wild-type and mutant proteins in the Saccharomyces cerevisiae strain PHY14, which lacks endogenous siderophore transporters, was confirmed by Western blotting. TAFC transport assays using (55)Fe-labeled TAFC and growth assays with Fe-TAFC as the sole iron source identified alanine 125, tyrosine 577, loop 3, and the second half of loop 7 (Loop7Del2) as crucial for function, since their substitution or deletion abrogated uptake completely. Wild-type MirB transported ferricrocin and coprogen as well as TAFC but not ferrichrysin. MirB was localized by fluorescence microscopy using antisera raised against a MirB extracellular loop peptide. Immunofluorescence microscopy showed that in yeast, wild-type MirB had a punctate distribution under the plasma membrane, as did the A125D and Y577A strains, indicating that the defect in transport of these mutants was unlikely to be due to mislocalization or degradation. MirB immunolocalization in A. fumigatus showed that the transporter was found in vesicles which cycled between the cytoplasm and the plasma membrane and was concentrated at the hyphal tips. The location of MirB was not influenced by the presence of the siderophore TAFC but was sensitive to internal iron stores.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Proteínas de Transporte de Membrana
/
Aspergillus fumigatus
/
Proteínas Fúngicas
/
Sideróforos
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Eukaryot Cell
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2012
Tipo del documento:
Article
País de afiliación:
Canadá