Trafficking of lysosomal cathepsin B-green fluorescent protein to the surface of thyroid epithelial cells involves the endosomal/lysosomal compartment.
Cathepsin B, a lysosomal cysteine proteinase, is involved in limited proteolysis of thyroglobulin with thyroxine liberation at the apical surface of thyroid epithelial cells. To analyze the trafficking of lysosomal enzymes to extracellular locations of thyroid epithelial cells, we have expressed a chimeric protein consisting of rat cathepsin B and green fluorescent protein. Heterologous expression in CHO cells validated the integrity of the structural motifs of the chimeric protein for targeting to endocytic compartments. Homologous expression, colocalization and transport experiments with rat thyroid epithelial cell lines FRT or FRTL-5 demonstrated the correct sorting of the chimeric protein into the lumen of the endoplasmic reticulum, and its subsequent transport via the Golgi apparatus and the trans-Golgi network to endosomes and lysosomes. In addition, the chimeras were secreted as active enzymes from FRTL-5 cells in a thyroid-stimulating-hormone-dependent manner. Immunoprecipitation experiments after pulse-chase radiolabeling showed that secreted chimeras lacked the propeptide of cathepsin B. Thus, the results suggest that cathepsin B is first transported to endosomes/lysosomes from where its matured form is retrieved before being secreted, supporting the view that endosome/lysosome-derived cathepsin B contributes to the potential of extracellular proteolysis in the thyroid.
Linke
Martin
M
Institut für Zellbiologie and Bonner Forum Biomedizin, Universität Bonn, Ulrich-Haberland-Str. 61a, D-53121 Bonn, Germany.
Herzog
Volker
V
Brix
Klaudia
K
D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D004847:Epithelial Cells / Q000201:enzymology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013961:Thyroid Gland / Q000166:cytology / Q000201:enzymology*
D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002401:Cathepsin B