Random Mutagenesis of the Proton-coupled Folate Transporter (SLC46A1), Clustering of Mutations, and the Bases for Associated Losses of Function

Abstract

Loss-of-function mutations in the proton-coupled folate transporter (PCFT, SLC46A1) result in the autosomal recessive disorder, hereditary folate malabsorption (HFM). Identification and characterization of HFM mutations provide a wealth of information on the structure-function relationship of this transporter. In the current study, PCR-based random mutagenesis was employed to generate unbiased loss-of-function mutations of PCFT, simulating the spectrum of alterations that might occur in the human disorder. A total of 26 mutations were generated and 4 were identical to HFM mutations. Eleven were base deletion or insertion mutations that led to a frameshift and, along with similar HFM mutations, are predominantly localized to two narrow regions of the pcft gene at the 5-end. Base substitution mutations identified in the current study and HFM patients were largely distributed across the pcft gene. Elimination of the ATG initiation codon by a one-base substitution (G > A) did not result in a complete lack of translation at the same codon consistent with rare non-ATG translation initiation. Among six missense mutants evaluated, three mutant PCFTs were not detected at the plasma membrane, one mutation resulted in decreased binding to folate substrate, and one had a reduced rate of conformational change associated with substrate translocation. The remaining PCFT mutant had defects in both processes. These results broaden understanding of the regions of the pcft gene prone to base insertion and deletion and inform further approaches to the analysis of the structure-function of PCFT.The proton-coupled folate transporter (PCFT) 2 (SLC46A1) plays a key role in intestinal folate absorption and folate transport into the central nervous system (1). Loss-of-function mutations in the pcft gene lead to the rare autosomal recessive disorder, hereditary folate malabsorption (HFM) characterized by markedly reduced folate levels in blood and cerebrospinal fluid (1-4). A homozygous mutation in most cases or two compound heterozygous mutations in two cases have been identified in all subjects with the clinical diagnosis of HFM indicating that this disease is caused solely by alterations of the pcft gene (1, 2, 5-12).Sixteen different loss-of-function pcft mutations have been identified to date in HFM patients. Six result in drastic changes in predicted protein sequences (nonsense). p.Y362_G398del, occurred multiple times in unrelated families and is the result of skipping of exon 3 during RNA splicing (1, 9, 13). C66X introduces a stop codon at position 66 due to a two-base substitution (5). Four frameshift mutations, p. E9Gfs, p.G65Afs, C66Lfs and N68Kfs, are due to base deletions or insertions (2,8,10,12). Ten remaining mutations resulted in a single amino acid substitution in the PCFT protein (missense). Five mutations occurred at charged resides (p.R113C, p.R113S, p.R376W, p.R376Q, and p.D156Y) (2, 6, 7, 11), whereas the other five, p.G147R, p.S318R, p.A335D, p.G338R, and p.P425R, involved substitutions of a non-charged, with a ch...

The Major Facilitative Folate Transporters Solute Carrier 19A1 and Solute Carrier 46A1: Biology and Role in Antifolate Chemotherapy of Cancer

“…4). Mutation of Glu232 (Gly) decreased the rate of carrier translocation, whereas mutations at Ile304 (Phe) and Leu161 (Arg) decreased substrate affinities (Zhao et al, 2011b). Mutation of Pro425 to Arg resulted in decreased binding of MTX and other (anti)folate substrates; however, PMX binding was preserved .…”

“…Other residues implicated as functionally important to hPCFT transport include Leu161 (TMD4), Glu232 (TMD6), Ile304 (TMD8), and Pro425 (flanks TMD12) (Zhao et al, 2011b) (Fig. 4).…”

Inhibition of the Proton-Coupled Folate Transporter (PCFT-SLC46A1) by Bicarbonate and Other Anions

“…Zeocin (0.1 mg/ml) was added to the growth medium to maintain carrier expression in R1-11-RFC-6 cells and R1-11-PCFT-4 cells, while 0.3 mg/ml hygromycin was added to maintain R1-11-PCFT-h cells. Transient transfectants that express wild-type PCFT were generated by transfection of the pcft expression vector into R1-11 cells as described previously (Zhao et al, 2011b).…”

Functional roles of the A335 and G338 residues of the proton-coupled folate transporter (PCFT-SLC46A1) mutated in hereditary folate malabsorption

“…The functional consequences of point mutations in PCFT in HFM that result in amino acid substitutions have been characterized with the demonstration of defects in protein stability and trafficking, folate substrate binding, and/or alterations in the rates of oscillation between the conformational states of the carrier (11,14,21,22). These studies, along with site-directed mutagenesis at these and other residues, have provided insights into the structure/function of this transporter and its secondary structure (26,27,28,35,36,37).…”