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Lack of Plasma Membrane Targeting of a G172D Mutant Thiamine Transporter Derived from Rogers Syndrome Family

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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.

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, post-translational 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 [3H] thiamine uptake assay.

Results

The mutant THTR-1 protein was undetectable in transfected cells grown at 37°C but was readily expressed in transfected cells cultured at 28°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.

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Acknowledgments

We wish to thank Drs Y. C. Broder and O. Shenkar for technical assistance with cell staining and confocal microscopy, respectively; Ms. A. Cohen for technical assistance; Dr G. Spira for the use of laboratory equipment; Drs V. Labay and D. Kornizer for fruitful suggestions; and Dr L. H. Matherly for the human RFC-HA expression plasmid.

This work was partially supported by the Israeli Academy of Sciences and Juvenile Diabetes International Foundation to N. C. and by Technion internal grants to A. A.

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Author notes

  1. Nadine Cohen

    Present address: Department of Pharmacogenomics, R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey, 08869, USA

Authors and Affiliations

  1. 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, 7 Efron St. Bat Galim, Haifa, Israel, 31096

    Dana Baron, Nadine Cohen & Ami Aronheim

  2. Department of Biology, The Technion-Israel Institute of Technology, 32000, Haifa, Israel

    Yehuda G. Assaraf

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  1. Dana Baron
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  2. Yehuda G. Assaraf
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Correspondence to Ami Aronheim.

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Baron, D., Assaraf, Y.G., Cohen, N. et al. Lack of Plasma Membrane Targeting of a G172D Mutant Thiamine Transporter Derived from Rogers Syndrome Family. Mol Med 8, 462–474 (2002). https://doi.org/10.1007/BF03402026

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  • DOI: https://doi.org/10.1007/BF03402026

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