Probing the mechanism of the hamster mitochondrial folate transporter by mutagenesis and homology modeling.
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.
Perchiniak
Erin
E
Department of Pharmacology and Toxicology and Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Lawrence
Scott A
SA
Kasten
Shane
S
Woodard
B Ann
BA
Taylor
Shirley M
SM
Moran
Richard G
RG
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
D033681:Mitochondrial Membrane Transport Proteins; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D000226:Mitochondrial ADP, ATP Translocases; D005492:Folic Acid