Membrane Topology of the Murine Fatty Acid Transport Protein 1

Abstract

The murine fatty acid transport protein (FATP1) was identified in an expression cloning screen for proteins that facilitate transport of fatty acids across the plasma membranes of mammalian cells. Hydropathy analysis of this protein suggests a model in which FATP1 has multiple membrane-spanning domains. To test this model, we inserted a hemagglutinin epitope tag at the amino terminus or a FLAG tag at the carboxyl terminus of the FATP1 cDNA and expressed these constructs in NIH 3T3 cells. Both tagged constructs produce proteins of the expected molecular masses and are functional in fatty acid import assays. Indirect immunofluorescence studies with selective permeabilization conditions and protease protection studies of sealed membrane vesicles from cells expressing epitope-tagged FATP1 were performed. These experiments show that the extreme amino terminus of tagged FATP1 is oriented toward the extracellular space, whereas the carboxyl terminus faces the cytosol. Additionally, enhanced green fluorescent protein fusion constructs containing predicted membrane-associated or soluble portions of FATP1 were expressed in Cos7 cells and analyzed by immunofluorescence and subcellular fractionation. These experiments demonstrate that amino acids 1-51, 52-100, and 101-190 contain signals for integral association with the membrane, whereas residues 258 -313 and 314 -475 are only peripherally membrane-associated. Amino 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 FATP1 as a polytopic membrane protein with at least one transmembrane and multiple membrane-associated domains. This study provides the first experimental evidence for topology of a member of the family of plasma membrane fatty acid transport proteins.

Oligomerization of the Murine Fatty Acid Transport Protein 1

“…Compared with native FATP1, FATPnHA has similar activity for fatty acid uptake. As has been previously observed, FATPcFLAG is less efficient in promoting fatty acid uptake, possibly due to perturbation of protein conformation upon placement of the epitope tag at the carboxyl terminus (9). Cells co-expressing both FATPnHA and FATPcFLAG have total FATP levels comparable to cells expressing either construct alone and comparable levels of fatty acid uptake.…”

“…Experimental evidence for the membrane topology of FATP1 supports a topology model with one transmembrane domain near the amino terminus of the protein (9). The extreme amino terminus of the protein faces the extracellular space, and the carboxyl terminus faces the cytosol.…”

Keratinocyte-specific Expression of Fatty Acid Transport Protein 4 Rescues the Wrinkle-free Phenotype in Slc27a4/Fatp4 Mutant Mice

“…2 compose a family of transmembrane proteins that facilitate cellular long chain fatty acid uptake (1)(2)(3). The most widely expressed member of this family is FATP4 (4,5), which is encoded by Slc27a4 (solute carrier family 27 member 4 gene).…”

Advances in adipose tissue metabolism

“…Amino acid sequences in the cytosolic section of FATP-1 contain the AMP-binding motif that mediates acyl CoA-synthase activity. 53 The primary sequence of the murine fatty acid transport protein (FATP-1) is very similar to that of the multigene family of very long-chain (C20-C26) ACSs. 54 A constitutive interaction between FATP-1 and long-chain ACS-1 contributes to the efficient cellular uptake of LCFAs in adipocytes through vectorial acylation.…”