I-FABP expression alters the intracellular distribution of the BODIPY C16 fatty acid analog

Abstract

To investigate the structure–function relationships of intestinal fatty acid-binding protein (I-FABP) in cellular fatty acid (FA) trafficking, we compared the distribution of a fluorescent FA analog (BODIPY FL C16) in Cos-1 cells transiently transfected with the wild type protein (wt I-FABP) to that of a variant deleted of the alpha helical domain (HL I-FABP). In vector-only cells, BODIPY fluorescence was distributed throughout the cytoplasm. In the absence of added FA, wt I-FABP was found largely in the perinuclear region with some cytoplasmic staining as well. Addition of BODIPY FL C16 to transfected cells showed that the fluorescent FA was essentially completely colocalized with the protein in the cytoplasmic and perinuclear regions as well as in cytoplasmic clusters that are not observed in the absence of wt I-FABP. For HL I-FABP, the distribution of the protein in the absence of FA was diffusely cytoplasmic, in marked contrast to the wt protein. Addition of BODIPY led to less extensive colocalization than that observed for wt I-FABP. In particular, no localization to the perinuclear region was found. Organelle colocalization studies showed that both proteins colocalized with mitochondria and endoplasmic reticulum/golgi markers, but little with a lysosomal marker. The perinuclear localization for wt I-FABP and BODIPY did not show colocalization with any of the markers tested. Taken together, these results indicate that I-FABP binds FA in vivo and that the helical domain may be important for targeting I-FABP to a perinuclear domain but not, perhaps, to the endoplasmic reticulum, golgi apparatus or mitochondria.

The Role of Intestinal Fatty Acid Binding Proteins in Protecting Cells from Fatty Acid Induced Impairment of Mitochondrial Dynamics and Apoptosis

“…6B). The inverse relation between G(0) and N has been shown in previous reports (20). This observation suggested the possibility of the presence of heterogeneous species (possibly larger oligomers) in the clusters.…”

“…To answer this, we monitored the localization of EGFP-tagged IFABP in the HeLa cells and measured the value of D (diffusion coefficient) using FCS. IFABP was earlier reported to exhibit diffused cytosolic localization [20]. In the present study, while the FA ligand was unavailable, IFABP was found to be soluble in the cytoplasm with persistent cluster formation within the cytosol and around the perinuclear region, a condition that was not observed in cells expressing the empty EGFP vector.…”

“…Previously it was assumed that the short alpha helix present in the protein served as its ligand binding domain [68][69][70], but later it was found that in the intracellular environment, the helix-less IFABP (HL-IFABP) could also transfer its FA ligand [20]. It was also reported that the helix region helped the protein to transfer its ligand in the perinuclear region whereas the HL-IFABP was left diffused in the cytoplasm with its FA ligand [20]. Acid Binding Protein present in the protein helps it to fold and attain native structure properly [15,71].…”

“…IFABPs, like other FABPs, receive, import and ferry their lipid ligands to respective metabolic sites [17][18][19]. IFABP localizes in the peri-nuclear regions with less diffused cytoplasmic existence in the absence of its FA ligand [20]. Lipids are essential for several cellular functions, sources of cellular energy [21] and can modulate signaling pathways.…”

“…In all experiments, desired control groups received comparable volume of vehicles as treated groups. BODIPY FL C16 was added as 1 µM for 30 mins at 37 0 C as mentioned elsewhere [20].…”