FRET microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes

The fate of fluorescently labeled pre-nsL-TP (Cy3-pre-nsL-TP) microinjected into BALB/c 3T3 fibroblasts was investigated by confocal laser scanning mi croscopy, The protein exhibited a distinct punctate fluorescence pattern an d colocalized to a high degree with the immunofluorescence pattern for the peroxisomal enzyme acyl-CoA oxidase, Proteolytic removal of the C-terminal leucine of the putative peroxisomal targeting sequence (AKL) resulted in a diffuse cytosolic fluorescence. These results indicate that microinjected C y3-pre-nsL-TP is targeted to peroxisomes. The association of nsL-TP with pe roxisomal enzymes was investigated in cells by measuring fluorescence reson ance energy transfer (FRET) between the microinjected Cy3-pre-nsL-TP and Cy 5-labeled antibodies against the peroxisomal enzymes acyl-CoA oxidase, 3-ke toacyl-CoA thiolase, bifunctional enzyme, PMP70 and catalase, The technique of photobleaching digital imaging microscopy (pbDIM), used to quantitate t he FRET efficiency on a pixel-by-pixel basis, revealed specific association of nsL-TP with acyl-CoA oxidase, 3-ketoacyl-CoA thiolase and bifunctional enzyme in the peroxisomes, These observations were corroborated by subjecti ng a peroxisomal matrix protein fraction to affinity chromatography on Seph arose-immobilized pre-nsL-TP, Acyl-CoA oxidase was retained. These studies provide strong evidence for a role of nsL-TP in the regulation of peroxisom al fatty acid beta-oxidation, e.g. by facilitating the presentation of subs trates and/or stabilization of the enzymes.