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Trafficking of tail-anchored proteins: transport from the endoplasmic reticulum to the plasma membrane and sorting between surface domains in polarised epithelial cells.
- Source :
-
Journal of cell science [J Cell Sci] 2002 Apr 15; Vol. 115 (Pt 8), pp. 1689-702. - Publication Year :
- 2002
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Abstract
- Tail-anchored (TA) proteins, which are defined by an N-terminal cytosolic region and a C-terminal transmembrane domain (TMD), provide useful models for studying the role of the TMD in sorting within the exo-endocytic system. Previous work has shown that a short TMD is required to keep ER-resident TA proteins from escaping to downstream compartments of the secretory pathway. To investigate the role of the TMD in TA protein sorting, we used model constructs, which consisted of GFP linked at its C-terminus to the tail region of cytochrome b(5) with TMDs of differing length or hydrophobicity. Expression of these constructs in CV-1 cells demonstrated that the feature determining exit from the ER is hydrophobicity and that if exit occurs, at least a part of the protein reaches the cell surface. To investigate which pathway to the surface is followed by plasma-membrane-directed TA constructs, we expressed the TA constructs in polarised Madin Darby Canine Kidney (MDCK) cells. The constructs with 22 and 25 residue TMDs were localised basolaterally, but addition at the C-terminus of a 20-residue peptide containing an N-glycosylation site resulted in glycosylation-dependent relocation of approximately 50% of the protein to the apical surface. This result suggests that TA proteins may reach the basolateral surface without a signal or that our constructs contain a weak basolateral determinant that is recessive to the apical information carried by the glycan. To assess the effect of the TMDs of endogenous TA proteins, GFP was linked to the tails of syntaxin 3 and 4, which localise to the apical and basolateral surface, respectively, of MDCK cells. The two GFP fusion proteins showed a different surface distribution, which is consistent with a role for the two syntaxin TMDs in polarised sorting.
- Subjects :
- Amino Acid Sequence
Animals
Cadherins metabolism
Cell Line
Cytochromes b5 chemistry
Cytochromes b5 metabolism
Detergents chemistry
Detergents metabolism
Epithelial Cells cytology
Golgi Apparatus metabolism
Green Fluorescent Proteins
Luminescent Proteins genetics
Luminescent Proteins metabolism
Membrane Proteins genetics
Microscopy, Confocal
Molecular Sequence Data
Protein Structure, Tertiary
Qa-SNARE Proteins
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins metabolism
Sequence Alignment
Cell Membrane metabolism
Cell Polarity
Endoplasmic Reticulum metabolism
Epithelial Cells metabolism
Membrane Proteins metabolism
Protein Transport physiology
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9533
- Volume :
- 115
- Issue :
- Pt 8
- Database :
- MEDLINE
- Journal :
- Journal of cell science
- Publication Type :
- Academic Journal
- Accession number :
- 11950887
- Full Text :
- https://doi.org/10.1242/jcs.115.8.1689