Fatty acid transport proteins: a current view of a growing family.

Long-chain fatty acids (LCFAs) are a major caloric component of our diet and are key metabolites for energy generation and storage. Physiological uptake of LCFAs across cell membranes is a saturable and competable process occurring at low concentrations, indicative of protein-mediated transport. Fatty acid transport proteins are a family of transmembrane proteins that enhance LCFA uptake and are produced in all fatty acid-utilizing tissues. Here, we review our current understanding of the function, expression patterns and regulation and subcellular localization of this interesting family of proteins.

Sec24p and Iss1p function interchangeably in transport vesicle formation from the endoplasmic reticulum in Saccharomyces cerevisiae.

The Sec23p/Sec24p complex functions as a component of the COPII coat in vesicle transport from the endoplasmic reticulum. Here we characterize Saccharomyces cerevisiae SEC24, which encodes a protein of 926 amino acids (YIL109C), and a close homologue, ISS1 (YNL049C), which is 55% identical to SEC24. SEC24 is essential for vesicular transport in vivo because depletion of Sec24p is lethal, causing exaggeration of the endoplasmic reticulum and a block in the maturation of carboxypeptidase Y. Overproduction of Sec24p suppressed the temperature sensitivity of sec23-2, and overproduction of both Sec24p and Sec23p suppressed the temperature sensitivity of sec16-2. SEC24 gene disruption could be complemented by overexpression of ISS1, indicating functional redundancy between the two homologous proteins. Deletion of ISS1 had no significant effect on growth or secretion; however, iss1Delta mutants were found to be synthetically lethal with mutations in the v-SNARE genes SEC22 and BET1. Moreover, overexpression of ISS1 could suppress mutations in SEC22. These genetic interactions suggest that Iss1p may be specialized for the packaging or the function of COPII v-SNAREs. Iss1p tagged with His(6) at its C terminus copurified with Sec23p. Pure Sec23p/Iss1p could replace Sec23p/Sec24p in the packaging of a soluble cargo molecule (alpha-factor) and v-SNAREs (Sec22p and Bet1p) into COPII vesicles. Abundant proteins in the purified vesicles produced with Sec23p/Iss1p were indistinguishable from those in the regular COPII vesicles produced with Sec23p/Sec24p.

Identification of the major intestinal fatty acid transport protein.

While intestinal transport systems for metabolites such as carbohydrates have been well characterized, the molecular mechanisms of fatty acid (FA) transport across the apical plasmalemma of enterocytes have remained largely unclear. Here, we show that FATP4, a member of a large family of FA transport proteins (FATPs), is expressed at high levels on the apical side of mature enterocytes in the small intestine. Further, overexpression of FATP4 in 293 cells facilitates uptake of long chain FAs with the same specificity as enterocytes, while reduction of FATP4 expression in primary enterocytes by antisense oligonucleotides inhibits FA uptake by 50%. This suggests that FATP4 is the principal fatty acid transporter in enterocytes and may constitute a novel target for antiobesity therapy.

Transport function and regulation of mitochondrial uncoupling proteins 2 and 3.

Uncoupling protein 1 (UCP1) dissipates energy and generates heat by catalyzing back-flux of protons into the mitochondrial matrix, probably by a fatty acid cycling mechanism. If the newly discovered UCP2 and UCP3 function similarly, they will enhance peripheral energy expenditure and are potential molecular targets for the treatment of obesity. We expressed UCP2 and UCP3 in Escherichia coli and reconstituted the detergent-extracted proteins into liposomes. Ion flux studies show that purified UCP2 and UCP3 behave identically to UCP1. They catalyze electrophoretic flux of protons and alkylsulfonates, and proton flux exhibits an obligatory requirement for fatty acids. Proton flux is inhibited by purine nucleotides but with much lower affinity than observed with UCP1. These findings are consistent with the hypothesis that UCP2 and UCP3 behave as uncoupling proteins in the cell.

Existence of uncoupling protein-2 antigen in isolated mitochondria from various tissues.

Antibodies against Escherichia coli-expressed uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) were raised by operating the blotted proteins into the spleen of minipigs. The antisera reacted more intensively with the recombinant UCP2 and UCP3 than with uncoupling protein-1 (UCP1) isolated from brown adipose tissue. Moreover, anti-UCP2 and cross-reacting anti-UCP3 antibodies identified the presence of the UCP2/3 antigen in isolated mitochondria from rat heart, rat kidney, rat brain, rabbit epididymal white adipose tissue, hamster brown adipose tissue, and rabbit skeletal muscle. It has been concluded that UCP2 is expressed in these tissues (UCP3 in skeletal muscle); however their existence in mitochondria had not previously been demonstrated.

COPII subunit interactions in the assembly of the vesicle coat.

In vitro analysis of COPII vesicle formation in the yeast Saccharomyces cerevisiae has demonstrated the requirement for three cytosolic factors: Sec31p-Sec13p, Sec23p-Sec24p, and Sar1p. Convergent evidence suggests that the peripheral endoplasmic reticulum (ER) membrane protein Sec16p also represents an important component of the vesicle assembly apparatus: SEC16 interacts genetically with all five COPII genes; Sec16p binds to Sec23p and Sec24p, is found on ER-derived transport vesicles, and is required in vitro for the efficient release of ER-derived vesicle cargo. In this report, we demonstrate an important functional interaction between Sec16p and Sec31p. First, we map onto Sec31p binding regions for Sec16p, Sec23p, Sec24p, and Sec13p. Second, we show that a truncation mutant of Sec31p specifically defective for Sec16p binding is unable to complement a sec31Delta mutant and cannot rescue the secretion defect of a temperature-sensitive sec31 mutant at nonpermissive temperatures. We propose that Sec16p organizes the assembly of a coat that is stabilized both by the interaction of Sec31p with Sec23p and Sec24p and by the interaction of these three components with Sec16p.

Cloning and characterization of an uncoupling protein homolog: a potential molecular mediator of human thermogenesis.

We have identified a novel cDNA encoding a protein highly homologous to the mammalian brown fat uncoupling protein (UCP). Unlike the known UCP, which is expressed specifically in brown adipose tissue, the UCP homolog (UCPH) mRNA is expressed in a variety of tissues, with predominant expression in human white adipose tissue and skeletal muscle. In the white adipose tissue of ob/ob and db/db mice, the UCPH transcript is induced approximately fivefold relative to lean littermate controls. Expression of murine UCPH in yeast results in growth inhibition under conditions that require aerobic respiration, but does not affect growth under anaerobic conditions. Furthermore, UCPH expression in yeast causes a decrease in the mitochondrial membrane potential, as judged by staining with the potential-sensitive dye DiOC6. These observations suggest that UCPH, like UCP, uncouples oxidative phosphorylation. The possibility that the UCPH protein is an important mediator of human thermogenesis is discussed.

COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent regions of Sec16p.

Formation of COPII-coated vesicles at the endoplasmic reticulum (ER) requires assembly onto the membrane of five cytosolic coat proteins, Sec23p, Sec24p, Sec13p, Sec31p, and Sar1p. A sixth vesicle coat component, Sec16p, is tightly associated with the ER membrane and has been proposed to act as a scaffold for membrane association of the soluble coat proteins. We previously showed that Sec23p binds to the C-terminal region of Sec16p. Here we use two-hybrid and coprecipitation assays to demonstrate that the essential COPII protein Sec24p binds to the central region of Sec16p. In vitro reconstitution of binding with purified recombinant proteins demonstrates that the interaction of Sec24p with the central domain of Sec16p does not depend on the presence of Sec23p. However, Sec23p facilitates binding of Sec24p to Sec16p, and the three proteins can form a ternary complex in vitro. Truncations of Sec24p demonstrate that the N-terminal and C-terminal regions of Sec24p display different binding specificities. The C terminus binds to the central domain of Sec16p, whereas the N terminus of Sec24p binds to both the central domain of Sec16p and to Sec23p. These findings define binding to Sec16p as a new function for Sec24p and support the idea that Sec16p organizes assembly of the COPII coat.

Yeast SEC16 gene encodes a multidomain vesicle coat protein that interacts with Sec23p.

Temperature-sensitive mutations in the SEC16 gene of Saccharomyces cerevisiae block budding of transport vesicles from the ER. SEC16 was cloned by complementation of the sec16-1 mutation and encodes a 240-kD protein located in the insoluble, particulate component of cell lysates. Sec16p is released from this particulate fraction by high salt, but not by nonionic detergents or urea. Some Sec16p is localized to the ER by immunofluorescence microscopy. Membrane-associated Sec16p is incorporated into transport vesicles derived from the ER that are formed in an in vitro vesicle budding reaction. Sec16p binds to Sec23p, a COPII vesicle coat protein, as shown by the two-hybrid interaction assay and affinity studies in cell extracts. These findings indicate that Sec16p associates with Sec23p as part of the transport vesicle coat structure. Genetic analysis of SEC16 identifies three functionally distinguishable domains. One domain is defined by the five temperature-sensitive mutations clustered in the middle of SEC16. Each of these mutations can be complemented by the central domain of SEC16 expressed alone. The stoichiometry of Sec16p is critical for secretory function since overexpression of Sec16p causes a lethal secretion defect. This lethal function maps to the NH2-terminus of the protein, defining a second functional domain. A separate function for the COOH-terminal domain of Sec16p is shown by its ability to bind Sec23p. Together, these results suggest that Sec16p engages in multiple protein-protein interactions both on the ER membrane and as part of the coat of a completed vesicle.

SED4 encodes a yeast endoplasmic reticulum protein that binds Sec16p and participates in vesicle formation.

SEC16 is required for transport vesicle budding from the ER in Saccharomyces cerevisiae, and encodes a large hydrophilic protein found on the ER membrane and as part of the coat of transport vesicles. In a screen to find functionally related genes, we isolated SED4 as a dosage-dependent suppressor of temperature-sensitive SEC16 mutations. Sed4p is an integral ER membrane protein whose cytosolic domain binds to the COOH-terminal domain of Sec16p as shown by two-hybrid assay and coprecipitation. The interaction between Sed4p and Sec16p probably occurs before budding is complete, because Sed4p is not found in budded vesicles. Deletion of SED4 decreases the rate of ER to Golgi transport, and exacerbates mutations defective in vesicle formation, but not those that affect later steps in the secretory pathway. Thus, Sed4p is important, but not necessary, for vesicle formation at the ER. Sec12p, a close homologue of Sed4p, also acts early in the assembly of transport vesicles. However, SEC12 performs a different function than SED4 since Sec12p does not bind Sec16p, and genetic tests show that SEC12 and SED4 are not functionally interchangeable. The importance of Sed4p for vesicle formation is underlined by the isolation of a phenotypically silent mutation, sar1-5, that produces a strong ER to Golgi transport defect when combined with sed4 mutations. Extensive genetic interactions between SAR1, SED4, and SEC16 show close functional links between these proteins and imply that they might function together as a multisubunit complex on the ER membrane.