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 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 beta-oxidation, e.g. by facilitating the presentation of substrates and/or stabilization of the enzymes.
Wouters
F S
FS
Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, NL-3584 CH, Utrecht, The Netherlands.
Bastiaens
P I
PI
Wirtz
K W
KW
Jovin
T M
TM
D016475:3T3 Cells / Q000648:ultrastructure; D018528:ATP-Binding Cassette Transporters; D000102:Acetyl-CoA C-Acyltransferase; D042962:Acyl-CoA Oxidase; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002374:Catalase; D002451:Cell Compartmentation; D002846:Chromatography, Affinity; D004735:Energy Transfer; D005227:Fatty Acids / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D008565:Membrane Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008830:Microbodies / Q000737:chemistry / Q000201:enzymology*; D008853:Microscopy / Q000379:methods; D009097:Multienzyme Complexes; D010088:Oxidoreductases; D010940:Plant Proteins; D011485:Protein Binding
D018528:ATP-Binding Cassette Transporters; C489271:Abcd3 protein, mouse; D002352:Carrier Proteins; D005227:Fatty Acids; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D010940:Plant Proteins; C072087:sterol carrier proteins; D010088:Oxidoreductases; D002374:Catalase; D042962:Acyl-CoA Oxidase; D000102:Acetyl-CoA C-Acyltransferase