Basolateral sorting of the cation-dependent mannose 6-phosphate receptor in Madin-Darby canine kidney cells. Identification of a basolateral determinant unrelated to clathrin-coated pit localization signals.
In polarized Madin-Darby canine kidney (MDCK) cells, sorting of membrane proteins in the trans-Golgi network for basolateral delivery depends on the presence of cytoplasmic determinants that are related or unrelated to clathrin-coated pit localization signals. Whether these signals mediate basolateral protein sorting through common or distinct pathways is unknown. The cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains clathrin-coated pit localization signals that are necessary for endocytosis and lysosomal enzyme targeting. In this study, we have addressed the function of these signals in polarized sorting of the CD-MPR. A chimeric protein, made of the luminal domain of the influenza virus hemagglutinin fused to the transmembrane and cytoplasmic domains of the CD-MPR was stably expressed in MDCK cells. This chimera (HCD) is able to interact with the AP-1 Golgi-specific assembly proteins and is detected on the basolateral plasma membrane of MDCK cells where it is endocytosed. Deletion analysis and site-directed mutagenesis of the cytoplasmic domain of the CD-MPR indicate that HCD chimeras devoid of clathrin-coated pit localization signals are still transported to the basolateral membrane where they accumulate. A HCD chimera containing only the transmembrane domain and the 12 membrane-proximal amino acids of the CD-MPR cytoplasmic tail is also found on the basolateral membrane but is unable to interact with the AP-1 assembly proteins. However, the overexpression of this mutant results in partial apical delivery. It is concluded, therefore, that the basolateral transport of this chimera requires a saturable sorting machinery distinct from AP-1.
Distel
B
B
European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Federal Republic of Germany.
Bauer
U
U
Le Borgne
R
R
Hoflack
B
B
D000595:Amino Acid Sequence; D000818:Animals; D002412:Cations; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D004285:Dogs; D004705:Endocytosis; D007668:Kidney / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017527:Receptor, IGF Type 2 / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D018808:Transcription Factor AP-1 / Q000378:metabolism
D002412:Cations; D002966:Clathrin; D017527:Receptor, IGF Type 2; D018808:Transcription Factor AP-1