title: Lack of plasma membrane targeting of a G172D mutant thiamine transporter derived from Rogers syndrome family.
abstract: 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.
  
  
  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.
authors.affiliation: Department of Molecular Genetics, the Rappaport Institute for Research in the Medical Sciences and the B. Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Dana
authors.initials: D
authors.lastname: Baron
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
authors.forename: Nadine
authors.initials: N
authors.lastname: Cohen
authors.forename: Ami
authors.initials: A
authors.lastname: Aronheim
mesh_terms: D016475:3T3 Cells; D019943:Amino Acid Substitution; D000749:Anemia, Megaloblastic / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D021381:Protein Transport / Q000502:physiology; D013696:Temperature; D013831:Thiamine / Q000378:metabolism
chemical_list: D026901:Membrane Transport Proteins; C120043:SLC19A2 protein, human; C492292:Slc19a2 protein, mouse; D013831:Thiamine