Intracellular trafficking of thyroid peroxidase to the cell surface.
For thyroid hormone synthesis, thyroid peroxidase (TPO) molecules must be transported from the endoplasmic reticulum via the Golgi complex to be delivered at the cell surface to catalyze iodination of secreted thyroglobulin. Like other glycoproteins, TPO molecules in transit to the cell surface have the potential to acquire endoglycosidase H resistance as a consequence of Golgi-based modification of their N-linked carbohydrates, and measurement of the intracellular distribution of TPO has often relied on this assumption. To examine TPO surface distribution in thyrocyte cell lines, we prepared new antibodies against rat TPO. Antibody reactivity was first established upon expression of recombinant rat (r) TPO in 293 cells, which were heterogeneous for surface expression as determined by flow cytometry. By cell fractionation, surface rTPO fractionated distinctly from internal pools of TPO (that co-fractionate with calnexin), yet surface TPO molecules remained endoglycosidase H (endo H)-sensitive. Although the FRTL5 (and PC Cl3) rat thyrocyte cell line also exhibits almost no endo H-resistant TPO, much of the endogenous rTPO is localized to the cell surface by immunofluorescence. Similar results were obtained by fractionation of FRTL5 cell membranes on sucrose gradients. We conclude that in FRTL5 cells, a large fraction of rTPO is delivered to the plasma membrane yet does not acquire Golgi-type processing of its N-glycans. Rat and mouse thyroid tissue TPO also shows little or no endo H resistance, although cell fractionation still needs to be optimized for these tissues.
Kuliawat
Regina
R
Division of Endocrinology and Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Ramos-Castañeda
Jose
J
Liu
Youfang
Y
Arvan
Peter
P
D000818:Animals; D002241:Carbohydrates / Q000737:chemistry; D002384:Catalysis; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002469:Cell Separation; D002499:Centrifugation, Density Gradient; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006026:Glycoside Hydrolases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007453:Iodide Peroxidase / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D013954:Thyroglobulin / Q000378:metabolism; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
D002241:Carbohydrates; D018076:DNA, Complementary; D013395:Sucrose; D013954:Thyroglobulin; D007453:Iodide Peroxidase; D006026:Glycoside Hydrolases; D014357:Trypsin