Immunolocalization of thimet oligopeptidase in chicken embryonic fibroblasts

An antiserum was raised against chicken thimet oligopeptidase (TOP), and specific antibodies were isolated by immunoadsorption. The specificity of the antibodies was confirmed by immunoblot analysis with the cell extract of cultured chicken fibroblasts. Subcellular localization of TOP in normal and Rous sarcoma virus-transformed chicken embryonic fibroblasts was investigated by indirect immunofluorescence staining with affinity-purified, monospecific anti-TOP antibodies. In addition to the diffuse reaction expected for a cytosolic enzyme, a punctate, cytoplasmic localization was observed. The appearance of the organelles containing TOP did not correspond to that expected for secondary lysosomes, mitochondria, endoplasmic reticulum, or Golgi apparatus, but did resemble that of endosomes. When cells were allowed to internalize Lucifer yellow before fixation, some of the organelles containing TOP were observed to contain Lucifer yellow. Moreover, those organelles containing TOP were positively immunolabeled with the anti-transferrin receptor antibody. We conclude that part of the TOP in chicken embryonic fibroblasts is located in endosomes and discuss the implications of this.