Oligomeric Structure of the Human Reduced Folate Carrier: IDENTIFICATION OF HOMO-OLIGOMERS AND DOMINANT-NEGATIVE EFFECTS ON CARRIER EXPRESSION AND FUNCTION

Zhanjun Hou,Larry H Matherly

doi:10.1074/jbc.m807206200

Abstract

The ubiquitously expressed reduced folate carrier (RFC) is the major transport system for folate cofactors in mammalian cells and tissues. Previous considerations of RFC structure and mechanism were based on the notion that RFC monomers were sufficient to mediate transport of folate and antifolate substrates. The present study examines the possibility that human RFC (hRFC) exists as higher order homo-oligomers. By chemical cross-linking, transiently expressed hRFC in hRFC-null HeLa (R5) cells with the homobifunctional cross-linker 1,3-propanediyl bis-methanethiosulfonate and Western blotting, hRFC species with molecular masses of hRFC homo-oligomers were identified. Hemagglutinin- and Myc epitope-tagged hRFC proteins expressed in R5 cells were co-immunoprecipitated from both membrane particulate and surface-enriched membrane fractions, indicating that oligomeric hRFC is expressed at the cell surface. By co-expression of wild type and inactive mutant S138C hRFCs, combined with surface biotinylation and confocal microscopy, a dominant-negative phenotype was demonstrated involving greatly decreased cell surface expression of both mutant and wild type carrier caused by impaired intracellular trafficking. For another hRFC mutant (R373A), expression of oligomeric wild type-mutant hRFC was accompanied by a significant and disproportionate loss of wild type activity unrelated to the level of surface carrier. Collectively, our results demonstrate the existence of hRFC homo-oligomers. They also establish the likely importance of these higher order hRFC structures to intracellular trafficking and carrier function.

Highlights

Folates are members of the B class of vitamins that are required for the synthesis of nucleotide precursors, serine, and methionine in one-carbon transfer reactions [1]
The present study examines the possibility that human reduced folate carrier (RFC) exists as higher order homo-oligomers
Identification of Higher Order human RFC (hRFC) Structures by Crosslinking—Chemical cross-linking is a powerful method for establishing proximities of proteins in their native state [42] and has been extensively used to study quaternary structures of membrane proteins [15]

Summary

Oligomeric Structure of the Human Reduced Folate Carrier

IDENTIFICATION OF HOMO-OLIGOMERS AND DOMINANT-NEGATIVE EFFECTS ON CARRIER EXPRESSION AND FUNCTION*. The ubiquitously expressed reduced folate carrier (RFC) is the major transport system for folate cofactors in mammalian cells and tissues. By co-expression of wild type and inactive mutant S138C hRFCs, combined with surface biotinylation and confocal microscopy, a dominant-negative phenotype was demonstrated involving greatly decreased cell surface expression of both mutant and wild type carrier caused by impaired intracellular trafficking. Our results demonstrate the existence of hRFC homo-oligomers They establish the likely importance of these higher order hRFC structures to intracellular trafficking and carrier function. The ubiquitously expressed reduced folate carrier (RFC) is widely considered to be the major transport system for folate co-factors in mammalian cells and tissues [3, 4]. A growing literature suggests that quaternary structure involving the formation of higher order oligomers (e.g. dimers, tetramers, etc.) is commonly an important feature of the structure and function of

Human Reduced Folate Carrier Oligomers

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION