Identifying the Lipid-Protein Interface of the Proton-Coupled Folate Transporter using Hydrophobic Photoreactive Probes

Abstract

The Proton-Coupled Folate Transporter (PCFT) encoded by the gene SLC46A1 is the major pathway for uptake of folic acid in the intestine and into the brain. Mutations in the PCFT gene lead to hereditary folate malabsorption (HFM), a folate deficiency disease characterized by impaired intestinal folate absorption and transport of folate into the central nervous system. PCFT is also involved in the uptake of antifolates such as Pemetrexed by tumor cells. At the present time our knowledge about the structure/function relationships of PCFT are very limited. Our initial goal is to identify the lipid-protein interface of PCFT by identifying transmembrane segments labeled by hydrophobic photoreactive probes like 1-Azidopyrene (1-AP) and 2-[3H]diazofluorene ([3H]DAF). These probes partition efficiently (>95%) into the lipid bilayer and upon UV-irradiation form covalent linkages with close-by amino acids. 1-AP and [3H]DAF labeled peptides can then be identified by mass spectrometry or by automated Edman degradation coupled with radio-sequencing. To carry out these experiments requires large amounts (several mg) of PCFT protein. Out of three expression systems we have evaluated (mammalian, cell-free, and bacterial), expression in BL21 E. coli is so far the most promising. From preliminary labeling experiments we have confirmed the membrane insertion of PCFT: 1) PCFT membranes were washed with NaHCO3 and urea, and 2) With 1-AP labeled PCFT, we identified peptide fragments of PCFT using mass spectrometry. Future experiments will involve: 1) screening of additional bacterial expression host strains, 2) labeling of PCFT with [3H]DAF and identification of labeled peptides as well as individual labeled amino-acids using radio-sequencing. Our results will define the PCFT lipid-protein interface and therefore will help verify 1) the α-helical secondary structure of transmembrane segments and 2) the arrangement of transmembrane segments within the membrane-spanning domain.