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

• Published in: The EMBO journal Vol. 17; no. 24; pp. 7179 - 7189
• Main Authors: Wouters, Fred S., Bastiaens, Philippe I.H., Wirtz, Karel W.A., Jovin, Thomas M.
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 15.12.1998
• Subjects: 3-ketoacyl-CoA thiolase; 3T3 Cells - ultrastructure; Acetyl-CoA C-Acyltransferase; Acyl-CoA Oxidase; Animals; ATP-Binding Cassette Transporters; Biological Transport; Carrier Proteins - metabolism; Catalase; Cell Compartmentation; Chromatography, Affinity; Energy Transfer; Fatty Acids - metabolism; fluorescence resonance energy transfer; Image Processing, Computer-Assisted; Membrane Proteins; Mice; Mice, Inbred BALB C; Microbodies - chemistry; Microbodies - enzymology; Microscopy - methods; Multienzyme Complexes; Oxidoreductases; Plant Proteins; PMP70; Protein Binding; sterol carrier protein-2
• ISSN: 0261-4189; 1460-2075; 1460-2075
• DOI: 10.1093/emboj/17.24.7179

The fate of fluorescently labeled pre‐nsL‐TP (Cy3‐pre‐nsL‐TP) microinjected into BALB/c 3T3 fibroblasts was investigated by confocal laser scanning microscopy. The protein exhibited a distinct punctate fluorescence pattern and 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 Cy3‐pre‐nsL‐TP is targeted to peroxisomes. The association of nsL‐TP with peroxisomal enzymes was investigated in cells by measuring fluorescence resonance energy transfer (FRET) between the microinjected Cy3‐pre‐nsL‐TP and Cy5‐labeled antibodies against the peroxisomal enzymes acyl‐CoA oxidase, 3‐ketoacyl‐CoA thiolase, bifunctional enzyme, PMP70 and catalase. The technique of photobleaching digital imaging microscopy (pbDIM), used to quantitate the FRET efficiency on a pixel‐by‐pixel basis, revealed a specific association of nsL‐TP with acyl‐CoA oxidase, 3‐ketoacyl‐CoA thiolase and bifunctional enzyme in the peroxisomes. These observations were corroborated by subjecting a peroxisomal matrix protein fraction to affinity chromatography on Sepharose‐immobilized pre‐nsL‐TP. Acyl‐CoA oxidase was retained. These studies provide strong evidence for a role of nsL‐TP in the regulation of peroxisomal fatty acid β‐oxidation, e.g. by facilitating the presentation of substrates and/or stabilization of the enzymes.