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A tyrosine-based sorting signal is involved in connexin43 stability and gap junction turnover

Thomas, Marc
Zosso, Nathalie
Staub, Olivier
Published in Journal of cell science. 2003, vol. 116, no. Pt 11, p. 2213-22
Abstract The gap junction protein connexin43 is known to have a rapid turnover, involving degradation by both the proteasomal and lysosomal systems, but the structural features of connexin43 that govern these actions are not known. The connexin43 C-terminal sequence contains a proline-rich region corresponding to the consensus of a protein-protein interaction PY-motif (xPPxY), and an overlapping putative tyrosine-based sorting signal (Yxxphi; =hydrophobic), known to play a role in the intracellular trafficking of many membrane proteins. As both motifs may control turnover of connexin43, we used a combination of metabolic radiolabelling, immuno-precipitation and functional assays to determine the possible role of these motifs in controlling degradation of human connexin43 expressed in SKHep1 cells. Mutation V289D in the tyrosine-based sorting motif increased the steady-state pool of connexin43 by approximately 3.5-fold, while mutation P283L in the PY-motif produced a comparatively modest augmentation (1.7-fold). No additive effect was observed when the overlapping tyrosine was mutated. In pulse-chase experiments, the Y286A substitution increased the half-life of connexin43 from 2 to 6 hours, indicating that the increased steady-state levels reflected reduced protein degradation. Moreover, expression at the junctional membrane, as well as gap junction-mediated intercellular communication (GJC), were nearly abolished by lysosomal inhibitors and Brefeldin A in cells expressing wild-type connexin43, but were unaffected in the tyrosine mutant. These results provide strong evidence that the tyrosine-based motif of human connexin43 is a prime determinant controlling connexin43 stability, and consequently GJC, by targeting connexin43 for degradation in the endocytic/lysosomal compartment.
Keywords Carcinoma, HepatocellularCell Line, TumorConnexin 43/genetics/metabolismCysteine Endopeptidases/metabolismEndosomes/metabolismGap Junctions/metabolismHumansLiver NeoplasmsLysosomes/metabolismMultienzyme Complexes/metabolismMutagenesis/physiologyProteasome Endopeptidase ComplexProtein Sorting Signals/physiologyProtein Transport/physiologyTyrosine/metabolism
PMID: 12730291
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Research group Mucoviscidose et jonctions GAP (229)
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THOMAS, Marc et al. A tyrosine-based sorting signal is involved in connexin43 stability and gap junction turnover. In: Journal of cell science, 2003, vol. 116, n° Pt 11, p. 2213-22. doi: 10.1242/jcs.00440 https://archive-ouverte.unige.ch/unige:27056

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Deposited on : 2013-03-26

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