Transport, function, and sorting of lactase-phlorizin hydrolase in Madin-Darby canine kidney cells.
Lactase-phlorizin hydrolase (LPH), a small intestinal brush-border glycoprotein, is synthesized as a single chain precursor (pro-LPH, M(r) = 215,000-230,000) that undergoes cleavage to the final mature form (LPHm, M(r) = 160,000 in the human). In the human and pig small intestine as well as in the colon carcinoma cell line Caco-2, this cleavage takes place intracellularly prior to insertion into the brush-border membrane. To assess the role of proteolytic cleavage on the transport, function, and sorting of LPH a stable Madin-Darby canine kidney cell line was generated which expresses LPH (denoted as MDCK-ML). Biosynthetic labeling experiments demonstrated that the transport kinetics and posttranslational processing pattern of LPH in this cell line are similar to those in intestinal cells. Moreover, the enzymatic activity was found to be indistinguishable from that of brush-border LPH (LPHm). The sorting of LPH was studied by biosynthetic labeling of cells grown on filters followed by cell surface immunoprecipitation. Here, we could demonstrate that the cleaved LPHm molecule was predominantly found at the apical membrane, whereas complex glycosylated uncleaved pro-LPH (pro-LPHc) was targeted to both domains, the apical as well as the basolateral. In pulse-chase experiments at 20 degrees C pro-LPH was arrested in the trans-Golgi network, and cleavage to LPHm did not take place. By contrast, when the chase temperature was raised to 37 degrees C transport of pro-LPHc resumed, and cleavage to LPHm occurred. We conclude that the proteolytic cleavage of pro-LPHc to LPHm is a post-trans-Golgi network event and is most likely not implicated in the sorting of LPH by exposition of otherwise masked sorting elements.
Jacob
R
R
Institute of Microbiology, Heinrich Heine University of Düsseldorf, Federal Republic of Germany.
Brewer
C
C
Fransen
J A
JA
Naim
H Y
HY
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003110:Colonic Neoplasms; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000201:enzymology; D007421:Intestine, Small / Q000201:enzymology; D007668:Kidney; D007700:Kinetics; D010694:Lactase-Phlorizin Hydrolase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine; D014162:Transfection; D014407:Tumor Cells, Cultured
D011498:Protein Precursors; D011994:Recombinant Proteins; D010694:Lactase-Phlorizin Hydrolase