title: Probing the mechanism of the hamster mitochondrial folate transporter by mutagenesis and homology modeling.
abstract: The mitochondrial folate transporter (MFT) was previously identified in human and hamster cells. Sequence homology of this protein with the inner mitochondrial membrane transporters suggested a domain structure in which the N- and C-termini of the protein are located on the mitochondrial intermembrane-facing surface, with six membrane-spanning regions interspersed by two intermembrane loops and three matrix-facing loops. We now report the functional significance of insertion of the c-myc epitope into the intermembrane loops and of a series of site-directed mutations at hamster MFT residues highly conserved in orthologues. Insertional mutagenesis in the first predicted intermembrane loop eliminated MFT function, but the introduction of a c-myc peptide into the second loop was without effect. Most of the hamster MFT residues studied by site-directed mutagenesis were remarkably resilient to these mutations, except for R249A and G192E, both of which eliminated folate transport activity. Homology modeling, using the crystal structure of the bovine ADP/ATP carrier (AAC) as a scaffold, suggested a similar three-dimensional structure for the MFT and the AAC. An ion-pair interaction in the AAC thought to be central to the mechanism of membrane penetration by ADP is predicted by this homology model to be replaced by a pi-cation interaction in MFT orthologues and probably also in other members of the family bearing the P(I/L)W motif. This model suggests that the MFT R249A and G192E mutations both modify the base of a basket-shaped structure that appears to constitute a trap door for the flux of folates into the mitochondrial matrix.
authors.affiliation: Department of Pharmacology and Toxicology and Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Perchiniak
authors.forename: Scott A
authors.initials: SA
authors.lastname: Lawrence
authors.forename: Shane
authors.initials: S
authors.lastname: Kasten
authors.forename: B Ann
authors.initials: BA
authors.lastname: Woodard
authors.forename: Shirley M
authors.initials: SM
authors.lastname: Taylor
authors.forename: Richard G
authors.initials: RG
authors.lastname: Moran
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005492:Folic Acid / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D000226:Mitochondrial ADP, ATP Translocases; D005492:Folic Acid