Fluorescent labelling of membrane fatty acid transporter CD36 (SR-B2) in the extracellular loop

CONTEXT: Upon palmitate oversupply, membrane fatty acid-transporter CD36 (SR-B2) permanently translocates from endosomal storage to the sarcolemma, inducing lipotoxicity. CD36 translocation results from endosomal alkalinisation elicited by palmitate-induced disattachment of the cytoplasmic V(1)-subcomplex from the membrane-integrated V(0)-subcomplex of vacuolar-type H(+)-ATPase. OBJECTIVE: Develop a CD36 fluorescent labeling technique as initial step towards live cell imaging. METHODS: Three human CD36 (hCD36) mutants were constructed via insertion of a tetracysteine motif at different positions within the extracellular domain. Constructs were lentivirally transduced for subsequent CD36 labeling with fluorescein-arsenical hairpin-binder (FlAsH). Cell imaging was combined with V(0)/V(1) immunostaining and Western blotting. RESULTS: Transduction of hCD36-wildtype and mutants yielded corresponding proteins in HL-1 cardiomyocytes. Tetracysteine mutant-2 (hCD36-TC2) showed similar fatty acid uptake to wildtype. FlAsH staining revealed a speckled pattern reminiscent of endosomes. We found decreased V(1) co-localization with CD36 upon high-palmitate culturing. Conversely, V(0) consistently co-localized with CD36. CONCLUSION: hCD36-TC2 is a possible candidate for application of biarsenical dyes in live imaging studies pending further investigation. Our data is compatible with V(0)/V(1) disassembly in high-palmitate-treated cells.