A Role for the Proton-coupled Folate Transporter (PCFT-SLC46A1) in Folate Receptor-mediated Endocytosis

Rongbao Zhao,Sang Hee Min,Yanhua Wang,Estela Campanella,Philip S Low,I David Goldman

doi:10.1074/jbc.m807665200

Abstract

Recently, this laboratory identified a proton-coupled folate transporter (PCFT), with optimal activity at low pH. PCFT is critical to intestinal folate absorption and transport into the central nervous system because there are loss-of-function mutations in this gene in the autosomal recessive disorder, hereditary folate malabsorption. The current study addresses the role PCFT might play in another transport pathway, folate receptor (FR)-mediated endocytosis. FRalpha cDNA was transfected into novel PCFT(+) and PCFT(-) HeLa sublines. FRalpha was shown to bind and trap folates in vesicles but with minimal export into the cytosol in PCFT(-) cells. Cotransfection of FRalpha and PCFT resulted in enhanced folate transport into cytosol as compared with transfection of FRalpha alone. Probenecid did not inhibit folate binding to FR, but inhibited PCFT-mediated transport at endosomal pH, and blocked FRalpha-mediated transport into the cytosol. FRalpha and PCFT co-localized to the endosomal compartment. These observations (i) indicate that PCFT plays a role in FRalpha-mediated endocytosis by serving as a route of export of folates from acidified endosomes and (ii) provide a functional role for PCFT in tissues in which it is expressed, such as the choroid plexus, where the extracellular milieu is at neutral pH.

Highlights

Failure to absorb folates in the absence of this transporter in hereditary folate malabsorption (HFM) is expected
The current report addresses the hypothesis that proton-coupled folate transporter (PCFT) is an endosomal folate exporter and thereby plays a role in folate receptor (FR)␣mediated endocytosis [1, 2, 21, 22], that the ubiquitous expression of PCFT in mammalian tissues may be related to this function, and that loss of this function may be a basis for the low cerebrospinal fluid (CSF) folate levels in HFM
This study provides evidence that supports a role for PCFT in folate receptor-mediated endocytosis by serving as a route of export of folates from acidified endosomes into the cytoplasm

Summary

EXPERIMENTAL PROCEDURES

Chemicals—[3Ј,5Ј,7,9-3H]folic acid, [3Ј,5Ј,7,9-3H](6S)-5methylTHF (5-methyltetrahydrofolate), [3Ј,5Ј,7-3H(N)]MTX (methotrexate) and [3Ј,5Ј,7, 9-3H(N)](6S)-5-formylTHF (5-formyltetrahydrofolate) were purchased from Moravek Biochemicals (Brea, CA). FR␣-mediated Transport and Uptake into Cytoplasm versus Membrane Vesicles—Cells grown in 12-well plates were washed twice with acid buffer (pH 3.5) and once with ice-cold HBS (pH 8.0). The cells were incubated with 0.5 ml of pre-warmed HBS (pH 8.0) containing 50 nM tritiated folate substrates in the presence or absence of 1 ␮M nonlabeled folic acid at 37 °C for 1 h At this pH, transport directly across the plasma membrane mediated by PCFT is minimized and transport measured is due predominantly to FR␣-mediated endocytosis. Was no significant difference in uptake between R5-FR12 and R1-11-FR2 cells At this pH, transport directly across the plasma membrane mediated by PCFT, as assessed in FR␣-negative cells, was negligible. After completion of the uptake interval, cells were 500 and 540 nm was collected

RESULTS

Control

Findings

DISCUSSION