Biosynthesis of endotubin

An apical early endosomal glycoprotein from developing rat intestinal epithelial cells

Katrina Allen, K. Erden Gokay, Mitchell A. Thomas, Becky A. Speelman, Jean Wilson

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)367-373
Number of pages7
JournalBiochemical Journal
Volume330
Issue number1
StatePublished - Feb 15 1998

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Biosynthesis
Rats
Glycoproteins
Epithelial Cells
Membranes
Immunoprecipitation
Coprecipitation
Antibodies
Membrane Proteins
Monoclonal Antibodies
Antibody Binding Sites
Epitope Mapping
Proteins
Endosomes
Isoelectric Focusing
Molecular mass
Sorting
Immunoblotting
Washing
Labeling

ASJC Scopus subject areas

  • Biochemistry

Cite this

Biosynthesis of endotubin : An apical early endosomal glycoprotein from developing rat intestinal epithelial cells. / Allen, Katrina; Gokay, K. Erden; Thomas, Mitchell A.; Speelman, Becky A.; Wilson, Jean.

In: Biochemical Journal, Vol. 330, No. 1, 15.02.1998, p. 367-373.

Research output: Contribution to journalArticle

Allen, Katrina ; Gokay, K. Erden ; Thomas, Mitchell A. ; Speelman, Becky A. ; Wilson, Jean. / Biosynthesis of endotubin : An apical early endosomal glycoprotein from developing rat intestinal epithelial cells. In: Biochemical Journal. 1998 ; Vol. 330, No. 1. pp. 367-373.
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