Cell Biology of the Human Thiamine Transporter-1 (hTHTR1)

Abstract

The human thiamine transporter hTHTR1 is involved in the cellular accumulation of thiamine (vitamin B1) in many tissues. Thiamine deficiency disorders, such as thiamine-responsive megaloblastic anemia (TRMA), which is associated with specific mutations within hTHTR1, likely impairs the functionality and/or intracellular targeting of hTHTR1. Unfortunately, nothing is known about the mechanisms that control the intracellular trafficking or membrane targeting of hTHTR1. To identify molecular determinants involved in hTHTR1 targeting, we generated a series of hTHTR1 truncations fused with the green fluorescent protein and imaged the targeting and trafficking dynamics of each construct in living duodenal epithelial cells. Whereas the full-length fusion protein was functionally expressed at the plasma membrane, analysis of the truncated mutants demonstrated an essential role for both NH 2 -terminal sequence and the integrity of the backbone polypeptide for cell surface expression. Most notably, truncation of hTHTR1 within a region where several TRMA truncations are clustered resulted in intracellular retention of the mutant protein. Finally, confocal imaging of the dynamics of intracellular hTHTR1 vesicles revealed a critical role for microtubules, but not microfilaments, in hTHTR1 trafficking. Taken together, these results correlate hTHTR1 structure with cellular expression profile and reveal a critical dependence on hTHTR1 backbone integrity and microtubule-based trafficking processes for functional expression of hTHTR1.Thiamine (vitamin B1) is a water-soluble micronutrient that is essential for many cellular functions relating to growth and development. For example, thiamine pyrophosphate (the coenzyme form) is important for normal carbohydrate metabolism and energy production (1, 2). Thiamine is not synthesized by humans and other mammals and is obtained from dietary sources via absorption into intestinal epithelia. Deficiencies in cellular thiamine accumulation lead to cardiovascular and neurological disorders and are associated with the inherited condition of thiamine-responsive megaloblastic anemia (TRMA) 1 (1, 3-7). Recent molecular analyses have identified the protein product of the SLC19A2 gene in humans (chromosome 1q23.3) as the site of mutations that link with TRMA inheritance (2, 7-10). SLC19A2 encodes a saturable, high affinity human thiamine transporter known as hTHTR1 (2, 7-10), which is expressed in many tissues, including duodenal epithelium (11). The functional properties of hTHTR1 mimic the biochemical characteristics of thiamine uptake in intestinal preparations (reviewed in Refs. 12 and 13). The full-length hTHTR1 protein encodes a 497-amino acid polypeptide with 12 predicted transmembrane-spanning segments and cytoplasmic NH 2 -and COOH-terminal regions (2, 8). Several clinically identified mutations have been characterized in hTHTR1 (2, 7-10, 14), including ten prematurely truncated mutants, resulting from either point mutation, deletion, or insertion of nucleotides (reviewed in Refs. 7 and 15...

Targeting and Trafficking of the Human Thiamine Transporter-2 in Epithelial Cells

“…Furthermore, measurements of steady-state targeting of hTHTR2 in the presence of microtubule-disrupting agents, or a cytoplasmic dynein inhibitor, disrupted the polarized expression of the full-length hTHTR2 polypeptide at the apical membrane domain. Comparison of these results with data obtained previously for hRFC and hTHTR1 (16,17) underscore commonalities shared between all members of the SLC19A gene family in regard to the structural features essential for cell surface delivery, despite their divergent targeting to discrete plasma membrane domains.…”

“…To determine the level of hTHTR2 expression in the stable cell lines, we performed semi-quantitative RT-PCR (17). Functionality of hTHTR2 was determined by [ 3 H]thiamine uptake assay.…”

A C-terminal Region Dictates the Apical Plasma Membrane Targeting of the Human Sodium-dependent Vitamin C Transporter-1 in Polarized Epithelia

“…To achieve sufficient spatial and temporal resolution to track individual structures, we used a scanning video rate confocal microscope and an oil immersion objective to monitor hSVCT1-YFP dynamics in cover glass-attached HuTu-80 cells, a preparation more amenable for trafficking analyses than water immersion imaging of filter-grown MDCK or CaCo-2 cells (18,20). First, we generated a stable hSVCT1-YFP expressing HuTu-80 cell line by G418 selection (Fig.…”

“…The full-length hS-VCT1-YFP fusion protein and truncated hSVCT1-YFP constructs were generated using the specific primer combinations defined in Table I under PCR conditions outlined previously (18). PCR products and the EYFP-N1 vector were subsequently digested with the restriction enzymes HindIII and SacII, the products were gel-isolated and ligated together to generate in-frame fusion proteins with enhanced yellow fluorescent protein (YFP) fused to the C terminus of each construct.…”

Thiamine Responsive Megaloblastic Anemia in Two Female Siblings

“…8 Distinct mutations in SLC19A2 have been described in many families 9 and lead to either a lack of the protein product or an altered structure of the protein so that it is not properly targeted to its site of function on the cell membrane. 9,10 TRMA is characterized by anemia, deafness, and diabetes mellitus. Hematological features consist of the unique combination of megaloblastic changes and the ring sideroblast abnormality, with variable degrees of neutropenia and thrombocytopenia.…”