A family of fatty acid transporters conserved from mycobacterium to man

Abstract

Long chain fatty acids (LCFAs) are an important source of energy for most organisms. They also function as blood hormones, regulating key metabolic functions such as hepatic glucose production. Although LCFAs can diffuse through the hydrophobic core of the plasma membrane into cells, this nonspecific transport cannot account for the high affinity and specific transport of LCFAs exhibited by cells such as cardiac muscle, hepatocytes, and adipocytes. Transport of LCFAs across the plasma membrane is facilitated by fatty acid transport protein (FATP), a plasma membrane protein that increases LCFA uptake when expressed in cultured mammalian cells [Schaffer, J. E. & Lodish, H. F. (1994) Cell 79, 427-436]. Here, we report the identification of four novel murine FATPs, one of which is expressed exclusively in liver and another only in liver and kidney. Both genes increase fatty acid uptake when expressed in mammalian cells. All five murine FATPs have homologues in humans in addition to a sixth FATP gene. FATPs are found in such diverse organisms as Fugu rubripes, Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae, and Mycobacterium tuberculosis. The function of the FATP gene family is conserved throughout evolution as the C. elegans and mycobacterial FATPs facilitate LCFA uptake when overexpressed in COS cells or Escherichia coli, respectively. The identification of this evolutionary conserved fatty acid transporter family will allow us to gain a better understanding of the mechanisms whereby LCFAs traverse the lipid bilayer as well as yield insight into the control of energy homeostasis and its dysregulation in diseases such as diabetes and obesity.

Figure 1

Sequence alignment of FATP clones.

Figure 2

LCFA uptake assays. (A) COS cells were cotransfected using the DEAE-dextran method with the mammalian expression vectors pCDNA-CD2 either alone (Control) or in combination with one of the FATP-containing expression vectors (pCDNA-mmFATP1, pCDNA-mmFATP2, or pCMV-SPORT2-mmFATP5) as described in Materials and Methods. COS cells were gated on FSC and side scatter, and the results shown represent >10,000 cells. Cells exhibiting >300 CD2 fluorescence units (vertical line) representing ≈15% of all cells were deemed CD2 positive. (B) As in A, COS cells were cotransfected with pCDNA-CD2 either alone (Control) or in combination with one of the FATP-containing expression vectors (pCDNA-mmFATP1, pCDNA-mmFATP2, pCMV-SPORT2-mmFATP5, or pCDNA-ceFATPb). The mean BODIPY-FA fluorescence of the CD2-positive cells is plotted; results shown represent the average of three experiments, each consisting of >50,000 COS cells. Note that a logarithmic scale is used on the ordinate. (C) The full-length coding region of mtFATP (squares) and a control protein (TFE3; circles) were subcloned into the inducible, prokaryotic expression vector pET (Novagen). Expression was induced (solid symbols) with 1 mM isopropyl β-d-thiogalactoside for 1 hr or cells were left uninduced (open symbols). Data points were done in triplicate and counts were normalized to the number of bacteria as determined by OD₆₀₀.

Figure 3

Northern blot analysis of murine FATP expression was analyzed using poly(A) mRNA blots (CLONTECH). Probes for each of the FATPs were derived from the 3′ untranslated repeat regions of each gene and were <60% identical in sequence.

Figure 4

Complete and partial sequences for FATP genes from human, rat, mouse, puffer fish, D. melanogaster, C. elegans, S. cerevisiae, and M. tuberculosis were aligned using ClustalX. Based on these data, a phylogenetic tree was generated using TreeViewPPC. The bar indicates the number of substitutions per residue, i.e., 0.1 corresponds to a distance of 10 substitutions per 100 residues.