The Molecular Intersection of Folate and Thiamine Transport

Recent structural and functional studies have revealed an unexpected molecular connection between folate and thiamine transport systems in mammalian cells. This review examines the intricate relationship between these essential B vitamins, focusing on their transport mechanisms and the newly discovered role of the reduced folate carrier (RFC1/SLC19A1) in thiamine homeostasis.

Introduction

Folate (vitamin B9) and thiamine (vitamin B1) are essential nutrients that play critical roles in cellular metabolism. While traditionally viewed as having separate transport systems, recent research has revealed intriguing connections between their cellular uptake mechanisms, particularly through the reduced folate carrier (RFC1/SLC19A1).

Folate Transport Systems

The Reduced Folate Carrier (RFC1/SLC19A1)

RFC1 is ubiquitously expressed in tissues and serves as the primary transport system for reduced folates and antifolate drugs. It functions as an antiporter, exchanging folates for organic phosphate anions across the plasma membrane. The carrier shows high specificity for reduced folates, with a particular preference for 5-methyltetrahydrofolate (5-MTHF), the predominant form in plasma.

Proton-Coupled Folate Transporter (PCFT/SLC46A1)

PCFT is predominantly expressed in the gastrointestinal tract and operates optimally at acidic pH. Unlike RFC1, PCFT couples folate transport to proton influx, making it particularly suited for dietary folate absorption in the acidic microenvironment of the intestine.

Folate Receptors (FRs)

Folate receptors operate through receptor-mediated endocytosis and show equal affinity for both folate and its reduced derivatives. They play crucial roles in folate delivery to the brain and folate retention in the kidney.

The RFC1-Thiamine Connection

Structural Homology

Despite sharing approximately 40% sequence identity with thiamine transporters THTR-1 (SLC19A2) and THTR-2 (SLC19A3), RFC1 shows distinct substrate specificity. While it cannot transport thiamine itself, recent studies have revealed its ability to transport phosphorylated thiamine derivatives.

RFC1 as a Thiamine Pyrophosphate Antiporter

A significant discovery is RFC1’s role in transporting thiamine pyrophosphate (TPP), the active form of thiamine. The carrier exhibits high affinity for TPP, with a Ki only about four times higher than its Kt for methotrexate. This transport capability has important implications for cellular thiamine homeostasis

Quote from the study:

Although multiple cellular metabolites such as ATP, ADP, AMP, and NAD+ have been documented as the coupled substrates of SLC19A1, we showed with the structural and functional analyses that TPP would be the favorite compared to those commonly recognized ones.

According to the alternative access mechanism, SLC19A1 would cycle between the inward-facing and outward-facing conformations to carry its substrates across the cell membrane.

In our current structures, the extracellular (5-MTHF) and intracellular (TPP) substrates are bound to the identical site in SLC19A1, similar to that observed in some other antiporters.

It is plausible that cytosolic TPP could liberate 5-MTHF from the inward-facing SLC19A1 through competition under physiological conditions, and SLC19A would then adopt the outward-facing conformation for releasing TPP and binding extracellular 5-MTHF again. (R1)

Impact on Cellular Thiamine Metabolism

Cells with high RFC1 expression show reduced accumulation of TPP, suggesting that RFC1-mediated TPP export may serve as a regulatory mechanism for intracellular thiamine levels. This process appears to be particularly relevant under physiological thiamine concentrations (30-50 nM).

However, high level RFC1 expression substantially reduced accumulation of the active thiamin coenzyme, thiamin pyrophosphate (TPP). This decreased level of TPP, synthesized intracellularly from imported thiamin, resulted from RFC1-mediated efflux of TPP. This conclusion was supported by the following observations

(i) Efflux of intracellular TPP was increased in cells with high expression of RFC1.

(ii) Methotrexate inhibits TPP influx.

(iii) TPP competitively inhibits methotrexate influx.

(iv) Loading cells, which overexpress RFC1 to high levels of methotrexate to inhibit competitively RFC1-mediated TPP efflux, augment TPP accumulation.

(v) There was an inverse correlation between thiamin accumulation and RFC1 activity in cells grown at a physiological concentration of thiamin.

The modulation of thiamin accumulation by RFC1 in murine leukemia cells suggests that this carrier may play a role in thiamin homeostasis and could serve as a modifying factor in thiamin nutritional deficiency as well as when the high affinity thiamin transporter is mutated. (R3)

Genetic mutation rs1051266 in SLC19A1 is associated with Wernicke-Korsakoff syndrome

Thirty non-synonymous variants were identified in the discovery cohort and, after filtering, 23 were taken forward and genotyped in the case-control cohort. Of these SLC19A1:rs1051266:G was nominally associated with WKS.

SLC19A1 encodes the reduced folate carrier, a major transporter for physiological folate in plasma; rs1051266 is reported to impact folate transport. Thiamine pyrophosphate (TPP) efflux was significantly decreased in HEK293 cells, stably transfected with rs1051266:G, under thiamine deficient conditions when compared with the efflux from cells transfected with rs1051266:A (P = 5.7 × 10-11).

CONCLUSION This study provides evidence for the role of genetic variation in the SLC19A1 gene, which may contribute to the development of WKS in vivo through modulation of TPP transport in cells. (R4)

Molecular Mechanism of Transport

Substrate Recognition

Recent structural studies have revealed how RFC1 recognizes both folate and thiamine derivatives. The carrier’s binding pocket accommodates the negative charges of both folate and TPP, though through different molecular interactions. Two variant residues (Arg133 and Gln377 in RFC1) appear crucial for determining substrate specificity among SLC19 family members.

Antiporter Mechanism

RFC1 operates through an alternating access mechanism, cycling between inward-facing and outward-facing conformations. The transport process is driven by the asymmetric distribution of organic phosphates across the cell membrane, with high intracellular concentrations providing the driving force for folate uptake.