The data presented here strengthen the evidence that FAT/CD36 partition into lipid rafts and that LCFA uptake mediated by FAT/CD36 depends on this association.
First of all, we could show that LCFA uptake was critically dependent on the integrity of lipid rafts. Lipid rafts are cholesterol and sphingolipid enriched microdomains. Removal of raft lipids from cells leads to disruption of raft functions . Consistent with previous results on other cells [14–17] we could demonstrate that by decreasing cellular levels of cholesterol, LCFA uptake in COS cells was also inhibited by > 50%. Additionally, we could show that by decreasing ceramide synthesis with myriocin or depleting membrane sphingolipids using external sphingomyelinase we were able to lower LCFA uptake. Sphingolipid depletion was less dramatic, probably because it is more difficult to lower cellular sphingolipid levels than cholesterol levels. The effects of sphingolipid synthesis inhibition may also be more pleiotropic because there are many different kinds of sphingolipids (sphingomyelins and glycosphingolipids). Nevertheless, we found that plasma membrane GM1 levels were reduced after myriocin treatment, demonstrating that not only cholesterol but also sphingolipids modulate LCFA uptake.
Another important finding supporting raft association was that after cross-linking with antibodies FAT/CD36 co-patched with placental alkaline phosphatase (PLAP), a GPI-anchored raft protein, in living cells and segregated from patches formed by cross-linking of GFP-TMD that served as a non-raft marker. This assay has previously been used to monitor if proteins associate with lipid rafts at the cell [25, 39]. Antibody cross-linking of surface proteins leads to the formation of plasma membrane clusters that can be easily observed during light microscopy. It has been shown that co-patching is dependent on plasma membrane cholesterol, as patching is inhibited by cholesterol removal .
Antibody cross-linking increased DRM association of FAT/CD36 as was previously demonstrated for other raft proteins [25, 31]. Because a considerable amount of FAT/CD36 was also found in the soluble fraction after detergent treatment, FAT/CD36 is probably found (in steady state) in two membrane pools, one raft associated and another localized in the surrounding bilayer. How partitioning between these two pools is regulated is an open question, but interestingly it has been shown that FAT/CD36 can dimerize . Oligomerization of raft components is known to lead to increased raft affinity [37, 38]. Therefore this mechanism might be important to regulate raft association of FAT/CD36.
It has been previously hypothesized that the detergent soluble fraction might represent intracellular FAT/CD36 and that upon cholesterol depletion FAT/CD36 might be stuck in the secretory pathway to the plasma membrane . It has been described, that FAT/CD36 can indeed translocate between the plasma membrane and intracellular compartments . However, recent data from Covey et al  using immunofluorescence and flow cytometry convincingly show that in 3T3-L1 adipocytes cholesterol depletion inhibits uptake of LCFAs without affecting FAT/CD36 or caveolin-1 distribution within the cells, indicating that cholesterol levels regulate LFCA uptake via a pathway that does not involve altered surface localization of FAT/CD36. Our cross-linking data, which were obtained on living cells at 10°C, can explain these findings because under these conditions intracellular antigens are not accessible to antibodies. Therefore increased DRM association of FAT/CD36 (Figure 5) after cross-linking with antibodies likely represent a horizontal recruitment of plasma membrane non-raft associated FAT/CD36 into raft domains. The enrichment of DRM associated FAT/CD36 could only be detected in presence of co-expressed FATP4. Whether the FATP4 acitivity as acyl-CoA synthetase drives cellular fatty acid influx and, thus, the requirement for raft associated FAT/CD36 or whether FATP4 is actively involved in the raft constitution process remains to be elucidated. FATP4 has been originally described to by a major fatty acid transporter at the apical membrane of enterocytes  and it has been speculated that both proteins might cooperate at the plasma membrane in fatty acid uptake [5, 41]. However recent data could not support this view . FATP4 seems rather to be an enzyme for acyl-CoA activity at the ER level and therefore indirectly increases fatty acid uptake. Deletion of FATP4 resulted in a perinatal lethality with a phenotype reminiscent of lethal restrictive dermopathy . By histology the skin was characterized by a hyperproliferative hyperkeratosis with a disturbed epidermal barrier. Lipid analysis of the skin revealed an increased proportion of ceramides and cholesterol. Within the ceramide fraction very long chain fatty acid substitutes were significantly reduced . These finding suggests that FATP4 is involved in ceramide biosynthesis, possibly by providing CoA activity for very long chain fatty acids. It is intriguing to speculate that this protein might therefore have also impact on lipid raft integrity. Rafts are enriched in sphingolipids (that are made from ceramides) and their long chain fatty acid substitutes have been implicated to be important to connect the outer and the inner layer of the microdomain. FATP4 would therefore be indirectly involved in the fatty acid uptake process by providing the molecules for a functional lipid raft assembly. This could explain our results of an enhanced raft association of FAT/CD36 after overexpression of FATP4.
In general clustering of lipid rafts can be achieved by different mechanisms . Besides antibodies also other ligands (e.g. insulin), lectins or linker proteins have been discussed. Which would be a reasonable mechanism for FAT/CD36 clustering? It is conceivable that under physiological conditions the clustering of FAT/CD36 is mediated by binding of long chain fatty acids. It was indeed shown that the artificial, long chain fatty acid [3H]-sulfo-N-succinimidyl oleate (SSO) – when incubated with adipocytes – binds to FAT/CD36 with high affinity and is almost exclusively found in DRMs . Thus the concentration of fatty acids presented to the plasma membrane may regulate the rate of fatty acid uptake by providing a sufficient functional platform in the form of FAT/CD36-raft complexes.