Disruption of transport activity in a D93H mutant thiamine transporter 1, from a Rogers Syndrome family.

Rogers syndrome is an autosomal recessive disorder resulting in megaloblastic anemia, diabetes mellitus, and sensorineural deafness. The gene associated with this disease encodes for thiamine transporter 1 (THTR1), a member of the SLC19 solute carrier family including THTR2 and the reduced folate carrier (RFC). Using transient transfections into NIH3T3 cells of a D93H mutant THTR1derived from a Rogers syndrome family, we determined the expression, post-translational modification, plasma membrane targeting and thiamine transport activity. We also explored the impact on methotrexate (MTX) transport activity of a homologous missense D88H mutation in the human RFC, a close homologue of THTR1. Western blot analysis revealed that the D93H mutant THTR1 was normally expressed and underwent a complete N-glycosylation. However, while this mutant THTR1 was targeted to the plasma membrane, it was completely devoid of thiamine transport activity. Consistently, introduction into MTX transport null cells of a homologous D88H mutation in the hRFC did not result in restoration of MTX transport activity, thereby suggesting that D88 is an essential residue for MTX transport activity. These results suggest that the D93H mutation does not interfere with transporter expression, glycosylation and plasma membrane targeting. However, the substitution of this negatively charged amino acid (Asp93) by a positively charged residue (His) in an extremely conserved region (the border of transmembrane domain 2/intracellular loop 2) in the SLC19 family, presumably inflicts deleterious structural alterations that abolish thiamine binding and/or translocation. Hence, this functional characterization of the D93H mutation provides a molecular basis for Rogers syndrome.

Lack of plasma membrane targeting of a G172D mutant thiamine transporter derived from Rogers syndrome family.

BACKGROUND: Rogers syndrome, also known as thiamine responsive megaloblastic anemia (TRMA), is an autosomal recessive disorder resulting in megaloblastic anemia, diabetes mellitus and sensorineural deafness. The gene associated with Rogers syndrome encodes for a plasma membrane thiamine transporter, THTR-1, a member of the solute carrier family that includes its homologue THTR-2 and the reduced folate carrier. MATERIALS AND METHODS: Using transient expression of wild-type and a missense mutant THTR-1 protein, derived from a TRMA family, in different cell lines and immunodetection analysis, we determined the expression, posttranslational modification, and subcellular localization of the wild-type and G172D mutant THTR-1. The transport activity of the transfected THTR-1 proteins was measured using a [(3) H] thiamine uptake assay. RESULTS: The mutant THTR-1 protein was undetectable in transfected cells grown at 37 degrees C but was readily expressed in transfected cells cultured at 28 degrees C, thereby allowing for further biochemical and functional analysis. In contrast to its fully glycosylated wild-type mature protein, the mutant THTR-1 protein underwent only the initial stage of N-linked glycosylation. The failure to undergo a complete glycosylation resulted in the lack of plasma membrane targeting and confinement of the mutant THTR-1 to the Golgi and endoplasmic reticulum (ER) compartment. Consistently, either treatment with tunicamycin or substitution of the THTR-1 consensus N-glycosylation acceptor asparagine 63 with glutamine, abolished its glycosylation and plasma membrane targeting. CONCLUSIONS: Taken collectively, these results suggest that the G172D mutation presumably misfolded THTR-1 protein that fails to undergo a complete glycosylation, is retained in the Golgi-ER compartment and thereby cannot be targeted to the plasma membrane. Finally, transfection studies revealed that the mutant G172D THTR-1 failed to transport thiamine. This is the first molecular and functional characterization of a missense mutant THTR-1 derived from a family with Rogers syndrome.