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

Abstract

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.