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doi: 10.1242/10.1242/jcs.00440
Research Article |
1 Institute of Pharmacology and Toxicology, University of Lausanne, 1005
Lausanne, Switzerland
2 Department of Pediatrics, University Hospitals, 1211 Geneva, Switzerland
3 Department of Physiology, University of Geneva, 1211 Geneva, Switzerland
Authors for correspondence (e-mails:
olivier.staub{at}ipharm.unil.ch;
marc.chanson{at}hcuge.ch)
Accepted 17 February 2003
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 (Yxx
;
=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.
Key words: Connexins, Gap junctional communication, Trafficking, Degradation, Endocytosis
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