Topology of the yeast fatty acid transport protein Fat1p: mechanistic implications for functional domains on the cytosolic surface of the plasma membrane

The fatty acid transport protein (FATP) Fat1p in the yeast Saccharomyces cerevisiae functions in concert with acyl-coenzyme A synthetase (ACSL; either Faa1p or Faa4p) in vectorial acylation, which couples the transport of exogenous fatty acids with activation to CoA thioesters. To further define the role of Fat1p in the transport of exogenous fatty acids, the topological orientation of two highly conserved motifs [ATP/AMP and FATP/very long chain acyl CoA synthetase (VLACS)], the carboxyl 124 amino acid residues, which bind the ACSL Faa1p, and the amino and carboxyl termini within the plasma membrane were defined. T7 or hemagglutinin epitope tags were engineered at both amino and carboxyl termini, as well as at multiple nonconserved, predicted random coil segments within the protein. Six different epitope-tagged chimeras of Fat1p were generated and expressed in yeast; the sidedness of the tags was tested using indirect immunofluorescence and protease protection by Western blotting. Plasma membrane localization of the tagged proteins was assessed by immunofluorescence. Fat1p appears to have at least two transmembrane domains resulting in a N(in)-C(in) topology. We propose that Fat1p has a third region, which binds to the membrane and separates the highly conserved residues comprising the two halves of the ATP/AMP motif. The N(in)-C(in) topology results in the placement of the ATP/AMP and FATP/VLACS domains of Fat1p on the inner face of the plasma membrane. The carboxyl-terminal region of Fat1p, which interacts with ACSL, is likewise positioned on the inner face of the plasma membrane. This topological orientation is consistent with the mechanistic roles of both Fat1p and Faa1p or Faa4p in the coupled transport/activation of exogenous fatty acids by vectorial acylation.     

Fatty acid transport in Saccharomyces cerevisiae. Directed mutagenesis of FAT1 distinguishes the biochemical activities associated with Fat1p

The fatty acid transport protein Fat1p functions as a component of the long-chain fatty acid transport apparatus in the yeast Saccharomyces cerevisiae. Fat1p has significant homologies to the mammalian fatty acid transport proteins (FATP) and the very long-chain acyl-CoA synthetases (VLACS). In order to further understand the functional roles intrinsic to Fat1p (fatty acid transport and VLACS activities), a series of 16 alleles carrying site-directed mutations within FAT1 were constructed and analyzed. Sites chosen for the construction of amino acid substitutions were based on conservation between Fat1p and the mammalian FATP orthologues and included the ATP/AMP and FATP/VLACS signature motifs. Centromeric and 2 mu plasmids encoding mutant forms of Fat1p were transformed into a yeast strain containing a deletion in FAT1 (fat1Delta). For selected subsets of FAT1 mutant alleles, we observed differences between the wild type and mutants in 1) growth rates when fatty acid synthase was inhibited with 45 microm cerulenin in the presence of 100 microm oleate (C(18:1)), 2) levels of fatty acid import monitored using the accumulation of the fluorescent fatty acid 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-S-indacene-3-dodecanoic acid and [(3)H]oleate, 3) levels of lignoceryl (C(24:0)) CoA synthetase activities, and 4) fatty acid profiles monitored using gas chromatography/mass spectrometry. In most cases, there was a correlation between growth on fatty acid/cerulenin plates, the levels of fatty acid accumulation, very long-chain fatty acyl-CoA synthetase activities, and the fatty acid profiles in the different FAT1 mutants. For several notable exceptions, the fatty acid transport and very long-chain fatty acyl-CoA synthetase activities were distinguishable. The characterization of these novel mutants provides a platform to more completely understand the role of Fat1p in the linkage between fatty acid import and activation to CoA thioesters.     

Murine FATP alleviates growth and biochemical deficiencies of yeast fat1Delta strains.

Saccharomyces cerevisiae is an ideal model eukaryote for studying fatty-acid transport. Yeast are auxotrophic for unsaturated fatty acids when grown under hypoxic conditions or when the fatty-acid synthase inhibitor cerulenin is included in the growth media. The FAT1 gene encodes a protein, Fat1p, which is required for maximal levels of fatty-acid import and has an acyl CoA synthetase activity specific for very-long-chain fatty acids suggesting this protein plays a pivotal role in fatty-acid trafficking. In the present work, we present evidence that Fat1p and the murine fatty-acid transport protein (FATP) are functional homologues. FAT1 is essential for growth under hypoxic conditions and when cerulenin was included in the culture media in the presence or absence of unsaturated fatty acids. FAT1 disruptants (fat1Delta) fail to accumulate the fluorescent long-chain fatty acid fatty-acid analogue 4, 4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-do decanoic acid (C1-BODIPY-C12), have a greatly diminished capacity to transport exogenous long-chain fatty acids, and have very long-chain acyl CoA synthetase activities that were 40% wild-type. The depression in very long-chain acyl CoA synthetase activities were not apparent in cells grown in the presence of oleate. Additionally, beta-oxidation of exogenous long-chain fatty acids is depressed to 30% wild-type levels. The reduction of beta-oxidation was correlated with a depression of intracellular oleoyl CoA levels in the fat1Delta strain following incubation of the cells with exogenous oleate. Expression of either Fat1p or murine FATP from a plasmid in a fat1Delta strain restored these phenotypic and biochemical deficiencies. Fat1p and FATP restored growth of fat1Delta cells in the presence of cerulenin and under hypoxic conditions. Furthermore, fatty-acid transport was restored and was found to be chain length specific: octanoate, a medium-chain fatty acid was transported in a Fat1p- and FATP-independent manner while the long-chain fatty acids myristate, palmitate, and oleate required either Fat1p or FATP for maximal levels of transport. Lignoceryl CoA synthetase activities were restored to wild-type levels in fat1Delta strains expressing either Fat1p or FATP. Fat1p or FATP also restored wild-type levels of beta-oxidation of exogenous long-chain fatty acids. These data show that Fat1p and FATP are functionally equivalent when expressed in yeast and play a central role in fatty-acid trafficking.     

Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification.

The processes that govern the regulated transport of long-chain fatty acids across the plasma membrane are quite distinct compared to counterparts involved in the transport of hydrophilic solutes such as sugars and amino acids. These differences stem from the unique physical and chemical properties of long-chain fatty acids. To date, several distinct classes of proteins have been shown to participate in the transport of exogenous long-chain fatty acids across the membrane. More recent work is consistent with the hypothesis that in addition to the role played by proteins in this process, there is a diffusional component which must also be considered. Central to the development of this hypothesis are the appropriate experimental systems, which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both (i) exhibit saturable long-chain fatty acid transport at low ligand concentrations, (ii) have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus, and (iii) can be easily manipulated using the tools of molecular genetics. In both systems, central players in the process of fatty acid transport are fatty acid transport proteins (FadL or Fat1p) and fatty acyl coenzyme A (CoA) synthetase (FACS; fatty acid CoA ligase [AMP forming] [EC 6.2.1.3]). FACS appears to function in concert with FadL (bacteria) or Fat1p (yeast) in the conversion of the free fatty acid to CoA thioesters concomitant with transport, thereby rendering this process unidirectional. This process of trapping transported fatty acids represents one fundamental mechanism operational in the transport of exogenous fatty acids.     

Long-chain fatty acid transport in bacteria andyeast. Paradigms for defining the mechanism underlying this protein-mediated process

Protein-mediated transport of exogenous long-chain fatty acids across the membrane has been defined in a number of different systems. Central to understanding the mechanism underlying this process is the development of the appropriate experimental systems which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both [1] exhibit saturable long-chain fatty acid transport at low ligand concentration; [2] have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus; and [3] can be easily manipulated using the tools of molecular genetics. In E. coli, this process requires the outer membrane-bound fatty acid transport protein FadL and the inner membrane associated fatty acyl CoA synthetase (FACS). FadL appears to represent a substrate specific channel for long-chain fatty acids while FACS activates these compounds to CoA thioesters thereby rendering this process unidirectional. This process requires both ATP generated from either substrate-level or oxidative phosphorylation and the proton electrochemical gradient across the inner membrane. In S. cerevisiae, the process of long-chain fatty acid transport requires at least the membrane-bound protein Fat1p. Exogenously supplied fatty acids are activated by the fatty acyl CoA synthetases Faa1p and Faa4p but unlike the case in E. coli, there is not a tight linkage between transport and activation. Studies evaluating the growth parameters in the presence of long-chain fatty acids and long-chain fatty acid transport profiles of a fat1 strain support the hypothesis that Fat1p is required for optimal levels of long-chain fatty acid transport.     

Comparative biochemical studies of the murine fatty acid transport proteins (FATP) expressed in yeast

The fatty acid transport protein (FATP) family is a group of proteins that are predicted to be components of specific fatty acid trafficking pathways. In mammalian systems, six different isoforms have been identified, which function in the import of exogenous fatty acids or in the activation of very long-chain fatty acids. This has led to controversy as to whether these proteins function as membrane-bound fatty acid transporters or as acyl-CoA synthetases, which activate long-chain fatty acids concomitant with transport. The yeast FATP orthologue, Fat1p, is a dual functional protein and is required for both the import of long-chain fatty acids and the activation of very long-chain fatty acids; these activities intrinsic to Fat1p are separable functions. To more precisely define the roles of the different mammalian isoforms in fatty acid trafficking, the six murine proteins (mmFATP1-6) were expressed and characterized in a genetically defined yeast strain, which cannot transport long-chain fatty acids and has reduced long-chain acyl-CoA synthetase activity (fat1Delta faa1Delta). Each isoform was evaluated for fatty acid transport, fatty acid activation (using C18:1, C20:4, and C24:0 as substrates), and accumulation of very long-chain fatty acids. Murine FATP1, -2, and -4 complemented the defects in fatty acid transport and very long-chain fatty acid activation associated with a deletion of the yeast FAT1 gene; mmFATP3, -5, and -6 did not complement the transport function even though each was localized to the yeast plasma membrane. Both mmFATP3 and -6 activated C20:4 and C20:4, while the expression of mmFATP5 did not substantially increase acyl-CoA synthetases activities using the substrates tested. These data support the conclusion that the different mmFATP isoforms play unique roles in fatty acid trafficking, including the transport of exogenous long-chain fatty acids.     

FATP1 mediates fatty acid-induced activation of AMPK in 3T3-L1 adipocytes

Fatty acid transport proteins are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. FATP-dependent production of AMP was evaluated using FATP4 proteoliposomes, and fatty acid-dependent activation of AMP-activated protein kinase (AMPK) was assessed in 3T3-L1 adipocytes. Insulin-stimulated fatty acid influx (palmitate or arachidonate) into cultured adipocytes resulted in an increase in the phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase. Consistent with the activation of AMPK, palmitate uptake into 3T3-L1 adipocytes resulted in an increase in intracellular [AMP]/[ATP]. The fatty acid-induced increase in AMPK activation was attenuated in a cell line expressing shRNA targeting FATP1. Taken together, these results demonstrate that, in adipocytes, insulin-stimulated fatty acid influx mediated by FATP1 regulates AMPK and provides a potential regulatory mechanism for balancing de novo production of fatty acids from glucose metabolism with influx of preformed fatty acids via phosphorylation of acetyl-CoA carboxylase.     

Characterization of the Acyl-CoA synthetase activity of purified murine fatty acid transport protein 1

Fatty acid transport protein 1 (FATP1) is an approximately 63-kDa plasma membrane protein that facilitates the influx of fatty acids into adipocytes as well as skeletal and cardiac myocytes. Previous studies with FATP1 expressed in COS1 cell extracts suggested that FATP1 exhibits very long chain acyl-CoA synthetase (ACS) activity and that such activity may be linked to fatty acid transport. To address the enzymatic activity of the isolated protein, murine FATP1 and ACS1 were engineered to contain a C-terminal Myc-His tag expressed in COS1 cells via adenoviral-mediated infection and purified to homogeneity using nickel affinity chromatography. Kinetic analysis of the purified enzymes was carried out for long chain palmitic acid (C16:0) and very long chain lignoceric acid (C24:0) as well as for ATP and CoA. FATP1 exhibited similar substrate specificity for fatty acids 16-24 carbons in length, whereas ACS1 was 10-fold more active on long chain fatty acids relative to very long chain fatty acids. The very long chain acyl-CoA synthetase activity of the two enzymes was comparable as were the Km values for both ATP and coenzyme A. Interestingly, FATP1 was insensitive to inhibition by triacsin C, whereas ACS1 was inhibited by micromolar concentrations of the compound. These data represent the first characterization of purified FATP1 and indicate that the enzyme is a broad substrate specificity acyl-CoA synthetase. These findings are consistent with the hypothesis that that fatty acid uptake into cells is linked to their esterification with coenzyme A.     

Enzymatic properties of purified murine fatty acid transport protein 4 and analysis of acyl-CoA synthetase activities in tissues from FATP4 null mice

Fatty acid transport protein 4 (FATP4) is an integral membrane protein expressed in the plasma and internal membranes of the small intestine and adipocyte as well as in the brain, kidney, liver, skin, and heart. FATP4 has been hypothesized to be bifunctional, exhibiting both fatty acid transport and acyl-CoA synthetase activities that work in concert to mediate fatty acid influx across biological membranes. To determine whether FATP4 is an acyl-CoA synthetase, the murine protein was engineered to contain a C-terminal FLAG epitope tag, expressed in COS1 cells via adenovirus-mediated infection and purified to near homogeneity using alpha-FLAG affinity chromatography. Kinetic analysis of the enzyme was carried out for long chain (palmitic acid, C16:0) and very long chain (lignoceric acid, C24:0) fatty acids as well as for ATP and CoA. FATP4 exhibited substrate specificity for C16:0 and C24:0 fatty acids with a V(max)/K(m) (C16:0)/V(max)/K(m) (C24:0) of 1.5. Like purified FATP1, FATP4 was insensitive to inhibition by triacsin C but was sensitive to feedback inhibition by acyl-CoA. Although purified FATP4 exhibited high levels of palmitoyl-CoA and lignoceroyl-CoA synthetase activity, extracts from the skin and intestine of FATP4 null mice exhibited reduced esterification for C24:0, but not C16:0 or C18:1, suggesting that in vivo, defects in very long chain fatty acid uptake may underlie the skin disorder phenotype of null mice.     

Human very long-chain acyl-CoA synthetase and two human homologs: initial characterization and relationship to fatty acid transport protein

Several human genes with a high degree of homology to rat very long-chain acyl-CoA synthetase (rVLCS) and mouse fatty acid transport protein (mFATP) were identified. Full-length cDNA clones were obtained for three genes, and predicted amino acid sequences were generated. Initial characterization indicated that one gene was most likely hVLCS, the human ortholog of rVLCS. The other two (hVLCS-H1 and hVLCS-H2) were more closely related to rVLCS than to mFATP. Phylogenetic analysis of amino acid sequences confirmed that hVLCS-H1 and hVLCS-H2 were evolutionarily closer to VLCSs than FATPs. Alignment of predicted amino acid sequences of human, rat and mouse VLCSs and FATPs revealed the existence of two highly conserved motifs. While one motif is also present in long-chain acyl-CoA synthetases, the other serves to distinguish the VLCS/FATP family from the long-chain synthetase family. Elucidation of the biochemical functions of all VLCS/FATP family members should provide new insights into cellular fatty acid metabolism.     

Fatty acid transport proteins

PURPOSE OF REVIEW: Fatty acid transport proteins are a family of proteins involved in fatty acid uptake and activation. This review summarizes recent progress in elucidating the function of fatty acid transport proteins. RECENT FINDINGS: Recent experiments clearly establish FATP1 as a regulated fatty acid transporter in both adipose tissue and muscle with important roles in energy homeostasis, thermogenesis and insulin resistance. Knockout of FATP5 in mice show it to be a bifunctional protein required for both hepatic fatty acid uptake and bile acid reconjugation. The most striking phenotype of FATP4 deletion is a defect in skin homeostasis, which may be due to its very long chain acyl-coenzyme A synthetase activity. Fatty acid transport proteins are increasingly being recognized as multifunctional proteins that can mediate the uptake of fatty acids as well as catalyze the formation of coenzyme A derivatives using long-chain and very-long chain fatty acids, bile acids and bile acid precursors as substrates. SUMMARY: Modulation of fatty acid transport protein function can result in altered energy homeostasis and insulin sensitivity, defective skin homeostasis, and altered bile acid metabolism. Both fatty acid uptake and enzymatic activity of fatty acid transport proteins likely contribute to these phenotypes. Future studies are needed to better understand the molecular mechanism of fatty acid transport protein function and the physiological role of FATP2, FATP3, and FATP6.     

Human Fatty Acid Transport Protein 2a/Very Long Chain Acyl-CoA Synthetase 1 (FATP2a/Acsvl1) Has a Preference in Mediating the Channeling of Exogenous n-3 Fatty Acids into Phosphatidylinositol

The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M_r 70,000) and FATP2b (M_r 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14–C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation.     

Molecular aspects of fatty acid transport: mutations in the IYTSGTTGXPK motif impair fatty acid transport protein function.

The murine fatty acid transport protein (FATP) facilitates uptake of long chain fatty acids (LCFAs) when expressed in mammalian cells. FATP's sequence contains a highly conserved motif, IYTSGTTGXPK, also found in a number of proteins known to interact with ATP. To explore the role of this motif, we independently mutated the central serine (serine 250) and threonine (threonine 252) residues in this motif and assessed the effects of these mutations on FATP function. When expressed in fibroblasts, the FATP mutants demonstrated impaired LCFA import and impaired binding of [alpha-32P]8-azido-ATP (azido-ATP) compared with wild-type FATP. These results suggest that serine 250 and threonine 252 are critical for FATP function and that the mechanism of action of FATP involves nucleotide binding which is dependent on these residues.     

Membrane topology of the murine fatty acid transport protein 1

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.     

The fatty acid transport protein (FATP1) is a very long chain acyl-CoA synthetase

The primary sequence of the murine fatty acid transport protein (FATP1) is very similar to the multigene family of very long chain (C20-C26) acyl-CoA synthetases. To determine if FATP1 is a long chain acyl coenzyme A synthetase, FATP1-Myc/His fusion protein was expressed in COS1 cells, and its enzymatic activity was analyzed. In addition, mutations were generated in two domains conserved in acyl-CoA synthetases: a 6- amino acid substitution into the putative active site (amino acids 249-254) generating mutant M1 and a 59-amino acid deletion into a conserved C-terminal domain (amino acids 464-523) generating mutant M2. Immunolocalization revealed that the FATP1-Myc/His forms were distributed between the COS1 cell plasma membrane and intracellular membranes. COS1 cells expressing wild type FATP1-Myc/His exhibited a 3-fold increase in the ratio of lignoceroyl-CoA synthetase activity (C24:0) to palmitoyl-CoA synthetase activity (C16:0), characteristic of very long chain acyl-CoA synthetases, whereas both mutant M1 and M2 were catalytically inactive. Detergent-solubilized FATP1-Myc/His was partially purified using nickel-based affinity chromatography and demonstrated a 10-fold increase in very long chain acyl-CoA specific activity (C24:0/C16:0). These results indicate that FATP1 is a very long chain acyl-CoA synthetase and suggest that a potential mechanism for facilitating mammalian fatty acid uptake is via esterification coupled influx.     

Fatty acid transport proteins and insulin resistance

PURPOSE OF REVIEW: Disturbed fatty acid metabolism and homeostasis is associated with insulin resistance. The aim of this review, therefore, is to summarize recent developments relating to the relevance and importance of the fatty acid transport proteins (FATPs) in the aetiology of insulin resistance. In particular, the potential differences between the six members of the FATP family will be considered. RECENT FINDINGS: FATP1 knockout mice failed to develop insulin resistance associated with lipid infusion or a high-fat diet, as wild-type mice did. FATP1-mediated fatty acid uptake may cause intramuscular lipid accumulation leading to insulin resistance in muscle if the fatty acids are not oxidized. While mouse models demonstrated an absolute requirement for FATP4 for survival, they provided no direct evidence for a role of FATP4 in insulin resistance. However, expression of FATP4 in human adipose tissue was increased in obesity (independent of genetic factors). While other members of the FATP family have important roles in fatty acid metabolism, they have not been clearly linked to insulin resistance. FATP-mediated fatty acid uptake may be driven by intrinsic acyl-CoA synthase activity. SUMMARY: Any role in the development of insulin resistance is likely to be different for each member of the FATP family. So far, both FATP1 and FATP4 have been associated with parameters related to insulin resistance. Whether increased FATP-mediated fatty acid uptake is beneficial or detrimental may be dependent on the tissue in question and on the subsequent fate of the fatty acids. These issues remain to be resolved.     

Structure-function relationships in UCP1, UCP2 and chimeras: EPR analysis and retinoic acid activation of UCP2.

Uncoupling proteins (UCPs) are composed of three repeated domains of approximately 100 amino acids each. We have used chimeras of UCP1 and UCP2, and electron paramagnetic resonance (EPR), to investigate domain specific properties of these UCPs. Questions include: are the effects of nucleotide binding on proton transport solely mediated by amino acids in the third C-terminal domain, and are the amino acids in the first two domains involved in retinoic or fatty acid activation? We first confirmed that our reconstitution system produced UCP1 that exhibited known properties, such as activation by fatty acids and inhibition of proton transport by purine nucleotides. Our results confirm the observations reported for recombinant yeast that retinoic acid, but not fatty acids known to activate UCP1, activates proton transport by UCP2 and that this activation is insensitive to nucleotide inhibition. We constructed chimeras in which the last domains of UCP1 or UCP2 were switched and tested for activation by fatty acids or retinoic acid and inhibition by nucleotides. U1U2 is composed of mUCP1 (amino acids 1-198) and hUCP2 (amino acids 211-309). Fatty acids activated proton transport of U1U2 and GTP mediated inhibition. In the other chimeric construct U2U1, hUCP2 (amino acids 1-210) and mUCP1 (amino acids 199-307), retinoic acid still acted as an activator, but no inhibition was observed with GTP. Using EPR, a method well suited to the analysis of the structure of membrane proteins such as UCPs, we confirmed that UCP2 binds nucleotides. The EPR data show large structural changes in UCP1 and UCP2 on exposure to ATP, implying that a putative nucleotide-binding site is present on UCP2. EPR analysis also demonstrated changes in conformation of UCP1/UCP2 chimeras following exposure to purine nucleotides. These data demonstrate that a nucleotide-binding site is present in the C-terminal domain of UCP2. This domain was able to inhibit proton transport only when fused to the N-terminal part of UCP1 (chimera U1U2). Thus, residues involved in nucleotide inhibition of proton transport are located in the two first carrier motifs of UCP1. While these results are consistent with previously reported effects of the C-terminal domain on nucleotide binding, they also demonstrate that interactions with the N-terminal domains are necessary to inhibit proton transport. Finally, the results suggest that proteins such as UCP2 may transport protons even though they are not responsible for basal or cold-induced thermogenesis.     

Mouse fatty acid transport protein 4 (FATP4): characterization of the gene and functional assessment as a very long chain acyl-CoA synthetase

FATP4 (SLC27A4) is a member of the fatty acid transport protein (FATP) family, a group of evolutionarily conserved proteins that are involved in cellular uptake and metabolism of long and very long chain fatty acids. We cloned and characterized the murine FATP4 gene and its cDNA. From database analysis we identified the human FATP4 genomic sequence. The FATP4 gene was assigned to mouse chromosome 2 band B, syntenic to the region 9q34 encompassing the human gene. The open reading frame was determined to be 1929 bp in length, encoding a polypeptide of 643 amino acids. Within the coding region, the exon-intron structures of the murine FATP4 gene and its human counterpart are identical, revealing a high similarity to the FATP1 gene. The overall amino acid identity between the deduced murine and human FATP4 polypeptides is 92.2%, and between the murine FATP1 and FATP4 polypeptides is 60.3%. Northern analysis showed that FATP4 mRNA was expressed most abundantly in small intestine, brain, kidney, liver, skin and heart. Transfection of FATP4 cDNA into COS1 cells resulted in a 2-fold increase in palmitoyl-CoA synthetase (C16:0) and a 5-fold increase in lignoceroyl-CoA synthetase (C24:0) activity from membrane extracts, indicating that the FATP4 gene encodes an acyl-CoA synthetase with substrate specificity biased towards very long chain fatty acids.     

Fatty acid transport protein 1 and long-chain acyl coenzyme A synthetase 1 interact in adipocytes

The fatty acid transport proteins (FATP) and long-chain acyl coenzyme A synthetase (ACSL) proteins have been shown to play a role in facilitating long-chain fatty acid (LCFA) transport in mammalian cells under physiologic conditions. The involvement of both FATP and ACSL proteins is consistent with the model of vectorial acylation, in which fatty acid transport is coupled to esterification. This study was undertaken to determine whether the functions of these proteins are coordinated through a protein-protein interaction that might serve as a point of regulation for cellular fatty acid transport. We demonstrate for the first time that FATP1 and ACSL1 coimmunoprecipitate in 3T3-L1 adipocytes, indicating that these proteins form an oligomeric complex. The efficiency of FATP1 and ACSL1 coimmunoprecipitation is unaltered by acute insulin treatment, which stimulates fatty acid uptake, or by treatment with isoproterenol, which decreases fatty acid uptake and stimulates lipolysis. Moreover, inhibition of ACSL1 activity in adipocytes impairs fatty acid uptake, suggesting that esterification is essential for fatty acid transport. Together, our findings suggest that a constitutive interaction between FATP1 and ACSL1 contributes to the efficient cellular uptake of LCFAs in adipocytes through vectorial acylation.     

Mechanistic studies of the long chain acyl-CoA synthetase Faa1p from Saccharomyces cerevisiae

Long chain acyl-CoA synthetase (ACSL; fatty acid CoA ligase: AMP forming; EC 6.2.1.3) catalyzes the formation of acyl-CoA through a process, which requires fatty acid, ATP and coenzymeA as substrates. In the yeast Saccharomyces cerevisiae the principal ACSL is Faa1p (encoded by the FAA1 gene). The preferred substrates for this enzyme are cis-monounsaturated long chain fatty acids. Our previous work has shown Faa1p is a principal component of a fatty acid transport/activation complex that also includes the fatty acid transport protein Fat1p. In the present work hexameric histidine tagged Faa1p was purified to homogeneity through a two-step process in the presence of 0.1% eta-dodecyl-beta-maltoside following expression at 15 degrees C in Escherichia coli. In order to further define the role of this enzyme in fatty acid transport-coupled activation (vectorial acylation), initial velocity kinetic studies were completed to define the kinetic parameters of Faa1p in response to the different substrates and to define mechanism. These studies showed Faa1p had a Vmax of 158.2 nmol/min/mg protein and a Km of 71.1 microM oleate. When the concentration of oleate was held constant at 50 microM, the Km for CoA and ATP were 18.3 microM and 51.6 microM respectively. These initial velocity studies demonstrated the enzyme mechanism for Faa1p was Bi Uni Uni Bi Ping Pong.