Supramolecular dynamics of gap junctions

128

Gap junctions are dynamic plasma membrane domains, and their protein constituents, the connexins, have a high turnover rate in most tissue types. However, the molecular mechanisms involved in degradation of gap junctions have remained largely unknown. Here, we show that ubiquitin is strongly relocalized to connexin-43 (Cx43; also known as Gja1) gap junction plaques in response to activation of protein kinase C. Cx43 remained ubiquitylated during its transition to a Triton X-100-soluble state and along its trafficking to early endosomes. Following internalization, Cx43 partly colocalized with the ubiquitin-binding proteins Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate; also known as Hgs) and Tsg101 (tumor susceptibility gene 101). Depletion of Hrs or Tsg101 by small interfering RNA abrogated trafficking of Cx43 from early endosomes to lysosomes. Under these conditions, Cx43 was able to undergo dephosphorylation and deubiquitylation, locate to the plasma membrane and form functional gap junctions. Simultaneous depletion of Hrs and Tsg101 caused accumulation of a phosphorylated and ubiquitylated subpopulation of Cx43 in early endosomes and in hybrid organelles between partly degraded annular gap junctions and endosomes. Collectively, these data reveal a central role of early endosomes in sorting of ubiquitylated Cx43, and identify Hrs and Tsg101 as crucial regulators of trafficking of Cx43 to lysosomes.

Section: Discussionsupporting

confidence: 82%

Ubiquitylation of the gap junction protein connexin-43 signals its trafficking from early endosomes to lysosomes in a process mediated by Hrs and Tsg101

Leithe

Kjenseth

Sirnes

et al. 2009

128

“…Cx43 differs from many other transmembrane proteins by its high turnover rate. Both the lysosome and the ubiquitin proteasome machinery have been shown to be involved in the degradation process of Cx43 gap junctions (Larsen and Hai-Nan, 1978;Naus et al, 1993;Laing and Beyer, 1995;Laing et al, 1997;Musil et al, 2000;Rutz and Hulser, 2001). In most cases ubiquitylation is essential for proteasomal degradation.…”

Section: Nedd4 Binds All Detectable Isoforms Of Cx43mentioning

confidence: 99%

“…Degradation of Cx43 gap junctions involves both the lysosomal (Larsen and Hai-Nan, 1978;Naus et al, 1993;Laing et al, 1997) and the proteasomal pathways (Laing and Beyer, 1995;Laing et al, 1997;Musil et al, 2000;Rutz and Hulser, 2001), with ubiquitin playing a fundamental role in both degradation systems.…”

Section: Introductionmentioning

confidence: 99%

“…Cx43 contains a PY motif in its C-terminus, which is flanked by phosphorylation sites at Ser279 and Ser282 and is degraded in part by the ubiquitylation pathway (Laing and Beyer, 1995;Rutz and Hulser, 2001). This, and the fact that phosphorylation of Cx43 plays an important role in channel gating, suggests that Cx43 is also targeted by ubiquitin protein ligases in a similar form as the epithelial Na + channel proteins (Asher et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Ubiquitin protein ligase Nedd4 binds to connexin43 by a phosphorylation-modulated process

Leykauf

Salek

Bomke

et al. 2006

127

132

Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization.

“…The degradation of Cx43 has been shown to occur by the lysosomal (Laing et al, 1997;Larsen and Hai, 1978;Musil et al, 2000;Naus et al, 1993;Vaughan and Lasater, 1990) and the ubiquitin-proteasomal (Laing et al, 1997;Musil et al, 2000;Laing and Beyer, 1995;Rutz and Hulser, 2001) pathways, the relative contribution of which appears to be largely cell-type specific. It is likely that some of the proteasomal degradation occurs at the level of the endoplasmic reticulum (ER), as a quality control step, to remove poorly folded or oligomerised connexin polypeptides VanSlyke et al, 2000).…”

mentioning

confidence: 99%

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

Thomas

Zosso

Scerri

et al. 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 (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