Fatty acid transport protein-1 mRNA expression in skeletal muscle and in adipose tissue in humans

Fatty acid transporter protein (FATP)-1 mRNA expression was investigated in skeletal muscle and in subcutaneous abdominal adipose tissue of 17 healthy lean, 13 nondiabetic obese, and 16 obese type 2 diabetic subjects. In muscle, FATP-1 mRNA levels were higher in lean women than in lean men (2.2 +/- 0.1 vs. 0.6 +/- 0.2 amol/microg total RNA, P < 0.01). FATP-1 mRNA expression was decreased in skeletal muscle in obese women both in nondiabetic and in type 2 diabetic patients (P < 0.02 vs. lean women in both groups), and in all women there was a negative correlation with basal FATP-1 mRNA level and body mass index (r = -0.74, P < 0.02). In men, FATP-1 mRNA was expressed at similar levels in the three groups both in skeletal muscle (0.6 +/- 0.2, 0.6 +/- 0.2, and 0.8 +/- 0.2 amol/microg total RNA in lean, obese, and type 2 diabetic male subjects) and in adipose tissue (0.9 +/- 0.2 amol/microg total RNA in the 3 groups). Insulin infusion (3 h) reduced FATP-1 mRNA levels in muscle in lean women but not in lean men. Insulin did not affect FATP-1 mRNA expression in skeletal muscle in obese nondiabetic or in type 2 diabetic subjects nor in subcutaneous adipose tissue in any of the three groups. These data show a gender-related difference in the expression of the fatty acid transporter FATP-1 in skeletal muscle of lean individuals and suggest that changes in FATP-1 expression may not contribute to a large extent to the alterations in fatty acid uptake in obesity and/or type 2 diabetes.     

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.     

Regulation of cardiolipin biosynthesis by fatty acid transport protein-1 IN HEK 293 cells

Cardiolipin (CL) is a major phospholipid involved in energy metabolism mammalian mitochondria and fatty acid transport protein-1 (FATP-1) is a fatty acid transport protein that may regulate the intracellular level of fatty acyl-Coenzyme A's. Since fatty acids are required for oxidative phosphorylation via mitochondrial oxidation, we examined the effect of altering FATP-1 levels on CL biosynthesis. HEK-293 mock- and FATP-1 siRNA transfected cells or mock and FATP-1 expressing cells were incubated for 24 h with 0.1 mM oleic acid bound to albumin (1:1 molar ratio) then incubated for 24 h with 0.1 mM [1,3-³H]glycerol and radioactivity incorporated into CL determined. FATP-1 siRNA transfected cells exhibited reduced FATP-1 mRNA and increased incorporation of [1,3-³H]glycerol into CL (2-fold, p < 0.05) compared to controls indicating elevation in de novo CL biosynthesis. The reason for this was an increase in [1,3-³H]glycerol uptake and increase in activity and mRNA expression of the CL biosynthetic enzymes. In contrast, expression of FATP-1 resulted a reduction in incorporation of [1,3-³H]glycerol into CL (65%, p < 0.05) indicating reduced CL synthesis. [1,3-³H]Glycerol uptake was unaltered whereas activity of cytidine-5′-diphosphate-1,2-diacyl-sn-glycerol synthetase (CDS) and CDS-2 mRNA expression were reduced in FATP-1 expressing cells compared to control. In addition, in vitro CDS activity was reduced by exogenous addition of oleoyl-Coenzyme A. The data indicate that CL de novo biosynthesis may be regulated by FATP-1 through CDS-2 expression in HEK 293 cells.     

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.     

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.     

Characterization of a heart-specific fatty acid transport protein

Fatty acids are a major source of energy for cardiac myocytes. Changes in fatty acid metabolism have been implicated as causal in diabetes and cardiac disease. The mechanism by which long chain fatty acids (LCFAs) enter cardiac myocytes is not well understood but appears to occur predominantly by protein-mediated transport. Here we report the cloning, expression pattern, and subcellular localization of a novel member of the fatty acid transport protein (FATP) family termed FATP6. FATP6 is principally expressed in the heart where it is the predominant FATP family member. Similar to other FATPs, transient and stable transfection of FATP6 into 293 cells enhanced uptake of LCFAs. FATP6 mRNA was localized to cardiac myocytes by in situ hybridization. Immunofluorescence microscopy of FATP6 in monkey and murine hearts revealed that the protein is exclusively located on the sarcolemma. FATP6 was restricted in its distribution to areas of the plasma membrane juxtaposed with small blood vessels. In these membrane domains FATP6 also colocalizes with another molecule involved in LCFA uptake, CD36. These findings suggest that FATP6 is involved in heart LCFA uptake, in which it may play a role in the pathogenesis of lipid-related cardiac disorders.     

Differences in transport of fatty acids and expression of fatty acid transporting proteins in adipose tissue of obese black and white women

We have reported that the rate of de novo triglyceride (TG) synthesis by omental, but not subcutaneous, adipose tissue was higher in African-American women (AAW) than in Caucasian women (CAW). The purpose of this study was to explore the potential mechanisms underlying this increase. Toward that end, we determined the activities of key enzymes in the pathway of TG synthesis, the rates of uptake of fatty acids by adipocytes, mRNA and protein levels of the fatty acid-transporting proteins FAT/CD36 and FATP, and mRNA and protein levels of PPARgamma in omental fat of AAW and CAW. The results showed 1) no difference in the activity of phosphofructokinase, glycerol-3-phosphate dehydrogenase, or diacylglycerol acyltransferase; 2) a higher rate of fatty acid uptake by adipocytes of the AAW; 3) an increase in the mRNA and protein levels of CD36 and FATP4 in the fat of the AAW; and 4) an increase in the mRNA and protein levels of PPARgamma, which can stimulate the expression of CD36 and FATP. These results suggest that the increase in the transport of fatty acid, which is mediated by the overexpression of the transport proteins in the omental adipose tissue of the AAW, might contribute to the higher prevalence of obesity in AAW.     

Diet-induced insulin resistance in mice lacking adiponectin/ACRP30

Here we investigated the biological functions of adiponectin/ACRP30, a fat-derived hormone, by disrupting the gene that encodes it in mice. Adiponectin/ACRP30-knockout (KO) mice showed delayed clearance of free fatty acid in plasma, low levels of fatty-acid transport protein 1 (FATP-1) mRNA in muscle, high levels of tumor necrosis factor-alpha (TNF-alpha) mRNA in adipose tissue and high plasma TNF-alpha concentrations. The KO mice exhibited severe diet-induced insulin resistance with reduced insulin-receptor substrate 1 (IRS-1)-associated phosphatidylinositol 3 kinase (PI3-kinase) activity in muscle. Viral mediated adiponectin/ACRP30 expression in KO mice reversed the reduction of FATP-1 mRNA, the increase of adipose TNF-alpha mRNA and the diet-induced insulin resistance. In cultured myocytes, TNF-alpha decreased FATP-1 mRNA, IRS-1-associated PI3-kinase activity and glucose uptake, whereas adiponectin increased these parameters. Our results indicate that adiponectin/ACRP30 deficiency and high TNF-alpha levels in KO mice reduced muscle FATP-1 mRNA and IRS-1-mediated insulin signaling, resulting in severe diet-induced insulin resistance.     

Maternal micronutrients and omega 3 fatty acids affect placental fatty acid desaturases and transport proteins in Wistar rats

Adequate supply of LCPUFA from maternal plasma is crucial for fetal normal growth and development. The present study examines the effect of maternal micronutrients (folic acid and vitamin B₁₂) and omega 3 fatty acids on placental mRNA levels of fatty acid desaturases (Δ5 and Δ6) and transport proteins. Pregnant female rats were divided into 6 groups at 2 levels of folic acid both in the presence and absence of vitamin B₁₂. Both the vitamin B₁₂ deficient groups were supplemented with omega 3 fatty acid. Maternal vitamin B₁₂ deficiency reduced placental mRNA and protein levels of Δ5 desaturase, mRNA levels of FATP1 and FATP4 (p<0.05 for all) as compared to control while omega 3 fatty acid supplementation normalized the levels. Our data for the first time indicates that altered maternal micronutrients and omega 3 fatty acids play a key role in regulating fatty acid desaturase and transport protein expression in placenta.     

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.     

Fatty Acid Transport Protein 1 (FATP1) Localizes in Mitochondria in Mouse Skeletal Muscle and Regulates Lipid and Ketone Body Disposal

FATP1 mediates skeletal muscle cell fatty acid import, yet its intracellular localization and metabolic control role are not completely defined. Here, we examine FATP1 localization and metabolic effects of its overexpression in mouse skeletal muscle. The FATP1 protein was detected in mitochondrial and plasma membrane fractions, obtained by differential centrifugation, of mouse gastrocnemius muscle. FATP1 was most abundant in purified mitochondria, and in the outer membrane and soluble intermembrane, but not in the inner membrane plus matrix, enriched subfractions of purified mitochondria. Immunogold electron microscopy localized FATP1-GFP in mitochondria of transfected C2C12 myotubes. FATP1 was overexpressed in gastrocnemius mouse muscle, by adenovirus-mediated delivery of the gene into hindlimb muscles of newborn mice, fed after weaning a chow or high-fat diet. Compared to GFP delivery, FATP1 did not alter body weight, serum fed glucose, insulin and triglyceride levels, and whole-body glucose tolerance, in either diet. However, fatty acid levels were lower and β-hydroxybutyrate levels were higher in FATP1- than GFP-mice, irrespective of diet. Moreover, intramuscular triglyceride content was lower in FATP1- versus GFP-mice regardless of diet, and β-hydroxybutyrate content was unchanged in high-fat-fed mice. Electroporation-mediated FATP1 overexpression enhanced palmitate oxidation to CO2, but not to acid-soluble intermediate metabolites, while CO2 production from β-hydroxybutyrate was inhibited and that from glucose unchanged, in isolated mouse gastrocnemius strips. In summary, FATP1 was localized in mitochondria, in the outer membrane and intermembrane parts, of mouse skeletal muscle, what may be crucial for its metabolic effects. Overexpressed FATP1 enhanced disposal of both systemic fatty acids and intramuscular triglycerides. Consistently, it did not contribute to the high-fat diet-induced metabolic dysregulation. However, FATP1 lead to hyperketonemia, likely secondary to the sparing of ketone body oxidation by the enhanced oxidation of fatty acids.     

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.     

Fatty acid metabolism in adipocytes: functional analysis of fatty acid transport proteins 1 and 4

The role of fatty acid transport protein 1 (FATP1) and FATP4 in facilitating adipocyte fatty acid metabolism was investigated using stable FATP1 or FATP4 knockdown (kd) 3T3-L1 cell lines derived from retrovirus-delivered short hairpin RNA (shRNA). Decreased expression of FATP1 or FATP4 did not affect preadipocyte differentiation or the expression of FATP1 (in FATP4 kd), FATP4 (in FATP1 kd), fatty acid translocase, acyl-coenzyme A synthetase 1, and adipocyte fatty acid binding protein but did lead to increased levels of peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha. Both FATP1 and FATP4 kd adipocytes exhibited reduced triacylglycerol deposition and corresponding reductions in diacylglycerol and monoacylglycerol levels compared with control cells. FATP1 kd adipocytes displayed an approximately 25% reduction in basal (3)H-labeled fatty acid uptake and a complete loss of insulin-stimulated (3)H-labeled fatty acid uptake compared with control adipocytes. In contrast, FATP4 kd adipocytes as well as HEK-293 cells overexpressing FATP4 did not display any changes in fatty acid influx. FATP4 kd cells exhibited increased basal lipolysis, whereas FATP1 kd cells exhibited no change in lipolytic capacity. Consistent with reduced triacylglycerol accumulation, FATP1 and FATP4 kd adipocytes exhibited enhanced 2-deoxyglucose uptake compared with control adipocytes. These findings define unique and distinct roles for FATP1 and FATP4 in adipose fatty acid metabolism.     

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.     

FATP1 silence inhibits the differentiation and induces the apoptosis in chicken preadipocytes

FATP1 plays an important role in the trafficking of free fatty acids in adipocytes, however, its precise function and relationship with other fatty acid transporters all remain poorly understood. In this study, FATP1 gene silencing was induced by transfecting siRNA of target sequence into chicken preadipocytes, then the expression of FABP was found down-regulated while the expression of FAT was raised. In addition, differential inhibition of the cells was observed and the expressions of PPARγ and C/EBPα were found down-regulated. Moreover, the silencing also induced the down-regulation of FAS and inhibited the adipogenesis in adipocytes. Of specific interest here was that FATP1 silencing significantly improved the expressions and activities of cell apoptotic factors Caspases 3 and BCL2 associated X protein (Bax). Consequently, FATP1 deficiency prevented the differentiation while induced apoptosis in chicken preadipocytes.     

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.     

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.