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Research Article |
1 Consiglio Nazionale delle Ricerche Cellular and Molecular Pharmacology Center
and Department of Medical Pharmacology, University of Milan, Milan,
Italy
2 Faculty of Pharmacy, University of Catanzaro `Magna Graecia', Catanzaro,
Italy
* Present address: Centre for Study and Research on Obesity, Department of
Preclinical Sciences, L. Sacco Hospital, University of Milan, Italy
Author for correspondence (e-mail:
Nica{at}csfic.mi.cnr.it
)
Accepted 14 January 2002
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
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.
Key words: Apical and basolateral sorting, Cytochrome b(5), Madin Darby Canine Kidney cells, N-glycosylation, Syntaxins, Transmembrane domain
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