Abstract
The compartments involved in polarized exocytosis of membrane proteins are not well defined. In this study we hypothesized that newly synthesized polymeric immunoglobulin receptors are targeted from the trans-Golgi network to endosomes prior to their appearance on the basolateral cell surface of polarized Madin-Darby canine kidney cells. To examine this hypothesis, we have used an assay designed to measure the meeting of newly synthesized receptors with a selective population of apical or basolateral endosomes loaded with horseradish peroxidase. We found that in the course of basolateral exocytosis, the wild-type polymeric immunoglobulin receptor is targeted from the trans-Golgi network to apical and basolateral endosomes. Phosphorylation of a Ser residue in the cytoplasmic tail of the receptor is implicated in this process. The biosynthetic pathway of apically sorted polymeric immunoglobulin receptor mutants similarly traversed apical endosomes, raising the possibility that apical receptors are segregated from basolateral receptors in apical endosomes. The post-endocytic pathway of transcytosing and recycling receptors also passed through apical endosomes. Together, these observations are consistent with the possibility that the biosynthetic and endocytic routes merge into endosomes and justify a model suggesting that endosomal recycling processes govern polarized trafficking of proteins traveling in both pathways.
Highlights
Epithelial cells carry out a variety of vectorial transport and secretory processes that profoundly depend upon the polarized distribution of proteins and lipids on their cell surface
WGA-horseradish peroxidase (HRP)-fluorescein isothiocyanate (FITC), in contrast, produced more extended staining patterns, probably because it is concentrated into tubular extensions of sorting endosomes and/or because of its entering the tubular portion of apical recycling endosomal compartment (ARE) or CE residing underneath the apical plasma membrane (Fig. 2A)
Our findings essentially argue that in polarized Madin-Darby canine kidney (MDCK) cells: (a) The BFA-sensitive exocytic road taken by basolaterally sorted polymeric immunoglobulin receptor (pIgR) involves apical endosomes that are not early endosomes and basolateral early (sorting) endosomes (BEE). (b) Phosphorylated Ser726 in the cytoplasmic domain of pIgR is essential for receptor targeting from the trans-Golgi network (TGN) to endosomes; mutational inactivation of this Ser confers pIgR incorporation into a BFA-insensitive basolateral route that bypasses endosomes
Summary
Lectin-triticum vulgaris (wheat germ agglutinin (WGA)) coupled to horseradish peroxidase (HRP) and free HRP Type II were from Sigma. Human apo-transferrin (hTfn; Biological Industries Co, Beit hemeek, Israel) was loaded with iron as described [38]. Rabbit antiserum directed against a peptide in the cytoplasmic domain of the rat TGN resident protein, TGN-38 [39], was kindly provided by Dr G. Banting (University of Bristol, Bristol, UK) and was used at 1:500 and 1:100 dilution for Western blotting and immunofluorescence, respectively. Fab fragments derived from affinity purified guinea pig anti-rabbit SC polyclonal antibodies were a kind gift from Prof. K. Mostov (University of California, San Francisco) and were prepared as described previously [40]. Radioiodination of Fabs was performed according to the iodine monochloride protocol [40]
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