Abstract
Endosomes are the site of sorting of internalized receptors and ligands in all cell types and, in polarized cells, the apical endosomal compartment is involved in the selective trans-epithelial transport of immunoglobulins and growth factors. The biochemical composition of this specialized compartment remains largely unresolved. We have characterized a glycoprotein, called endotubin, that is located in the apical endosomal tubules of developing rat intestinal epithelial cells. A monoclonal antibody against endotubin recognizes a broad band of 55-60 kDa, which upon isoelectric focusing can be resolved into two bands, and a faint band of 140 kDa. Metabolic labelling followed by immunoprecipitation indicates that endotubin is synthesized as a 140 kDa precursor that is cleaved to the 55-60 kDa forms. High pH washing of endosomal membranes removes the 55-60 kDa forms from the membrane, whereas the high-molecular-mass form remains membrane associated and appears to be an integral membrane protein. Immunoblotting with a polyclonal antibody against the putative cytoplasmic tail of the protein identifies a 140 kDa band and a band of 74 kDa, presumably the cleavage product. Immunoprecipitation with antibodies against the 55-60 kDa form results in coprecipitation of a 74 kDa protein, and immunoprecipitation with antibody against the 74 kDa protein results in coprecipitation of the 55-60 kDa form. Epitope mapping of the monoclonal antibody binding site supports a proposed type I membrane protein orientation. We propose that endotubin is proteolytically processed into a heterodimer with the 55-60 kDa fragment remaining membrane-associated through a non-covalent association with the membrane-bound 74 kDa portion of the molecule.
Original language | English (US) |
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Pages (from-to) | 367-373 |
Number of pages | 7 |
Journal | Biochemical Journal |
Volume | 330 |
Issue number | 1 |
DOIs | |
State | Published - Feb 15 1998 |
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology