Membrane topology of the murine fatty acid transport protein 1

The murine fatty acid transport protein (FATP1) was identified in an expres sion cloning screen for proteins that facilitate transport of fatty acids a cross the plasma membranes of mammalian cells. Hydropathy analysis of this protein suggests a model in which FATP1 has multiple membrane-spanning doma ins. To test this model, we inserted a hemagglutinin epitope tag at the ami no terminus or a FLAG tag at the carboxyl terminus of the FATP1 cDNA and ex pressed these constructs in NIH 3T3 cells. Both tagged constructs produce p roteins of the expected molecular masses and are functional in fatty acid i mport assays. Indirect immunofluorescence studies with selective permeabili zation conditions and protease protection studies of sealed membrane vesicl es from cells expressing epitope-tagged FATP1 were performed. These experim ents show that the extreme amino terminus of tagged FATP1 is oriented towar d the extracellular space, whereas the carboxyl terminus faces the cytosol. Additionally, enhanced green fluorescent protein fusion constructs contain ing predicted membrane-associated or soluble portions of FATP1 were express ed in Cos7 cells and analyzed by immunofluorescence and subcellular fractio nation. These experiments demonstrate that amino acids 1-51, 52-100, and 10 1-190 contain signals for integral association with the membrane, whereas r esidues 258-313 and 314-475 are only peripherally membrane-associated. Amin o acid residues 191-257 and 476-646 do not direct membrane association and likely face the cytosol. Taken together, these data support a model of FATP 1 as a polytopic membrane protein with at least one transmembrane and multi ple membrane-associated domains. This study provides the first experimental evidence for topology of a member of the family of plasma membrane fatty a cid transport proteins.