Cytosolic Stress Reduces Degradation of Connexin43 Internalized from the Cell Surface and Enhances Gap Junction Formation and Function

Abstract

The protein constituents of gap junctions, connexins, have a rapid basal rate of degradation even after transport to the cell surface. We have used cell surface biotinylation to label gap junction-unassembled plasma membrane pools of connexin43 (Cx43) and show that their degradation is inhibited by mild hyperthermia, oxidative stress, and proteasome inhibitors. Cytosolic stress does not perturb endocytosis of biotinylated Cx43, but instead it seems to interfere with its targeting and/or transport to the lysosome, possibly by increasing the level of unfolded protein in the cytosol. This allows more Cx43 molecules to recycle to the cell surface, where they are assembled into long-lived, functional gap junctions in otherwise gap junction assembly-inefficient cells. Cytosolic stress also slowed degradation of biotinylated Cx43 in gap junction assembly-efficient normal rat kidney fibroblasts, and reduced the rate at which gap junctions disappeared from cell interfaces under conditions that blocked transport of nascent connexin molecules to the plasma membrane. These data demonstrate that degradation from the cell surface can be down-regulated by physiologically relevant forms of stress. For connexins, this may serve to enhance or preserve gap junction-mediated intercellular communication even under conditions in which protein synthesis and/or intracellular transport are compromised.

Connexins, cell motility, and the cytoskeleton

“…This F-actin binding protein, when silenced with siRNA, displayed impaired junctional coupling, increased GJ internalization, and targeting of Cx43 to degradative pathways [Butkevich et al, 2004]. The dependence of GJIC on internalization and subse-quent degradation has been shown elegantly by the group of Linda Musil [VanSlyke and Musil, 2005;Kelly et al, 2007]. Their data demonstrate that degradation from the cell surface can be down-regulated by physiologically relevant forms of stress.…”

The connexin43-interacting protein, CIP85, mediates the internalization of connexin43 from the plasma membrane

“…It appears that all of the identifi ed processes of internalization terminate in the lysosomal degradation of Cx43 (Guan and Ruch, 1996;Hesketh et al, 2010;Laing & Beyer, 1995;Laing et al, 1998;Laing et al, 1997;Leithe et al, 2006;Leithe et al, 2009;Lichtenstein et al, 2011;Murray et al, 1981;Naus et al, 1993;Qin et al, 2003;Sasaki & Garant, 1986;Simeckova et al, 2009;Thomas et al, 2003;Fong et al, 2012). However, there is also evidence that a portion of the Cx43 that is internalized may return to the plasma membrane by recycling mechanisms (Boassa et al, 2010;Gilleron et al, 2011;VanSlyke & Musil, 2005;Xie et al, 1997). Despite the growing body of knowledge regarding the processes of Cx43 internalization, the precise molecular mechanisms that target Cx43 for Figure 6.…”

“…Increased Cx43 turnover can rapidly decrease the amount of Cx43 present at the plasma membrane, which could affect GJIC and mechanotransduction-associated Cx43 activities (Burra et al, 2010;Gumpert et al, 2008;Leithe et al, 2009;Sirnes et al, 2008;VanSlyke & Musil, 2005). This could also affect the stabilization of cell adhesion complexes through interactions between Cx43 and proteins like ZO-1 and integrin (Carette et al, 2010;Gilleron et al, 2008;Mendoza-Naranjo et al, 2012;Sin et al, 2009).…”

“…Connexins are cotranslationally inserted into the ER membrane and transported to the Golgi apparatus, where most connexin proteins oligomerize into hexameric hemichannels (Segretain & Falk, 2004;VanSlyke and Musil, 2005;Das Sarma et al, 2002;Maza et al, 2005). These channels are transported to the plasma membrane, where they can either open and function as transmembrane hemichannels (Bargiotas et al, 2009;SillerJackson et al, 2008;Scemes et al, 2009) or dock with hemichannels from adjacent cells to form intercellular gap junction channels that aggregate into large plaques at the plasma membrane (Goodenough et al, 1996;Wei et al, 2004).…”

“…Furthermore, it has been shown that gap junctions, which are aggregated in plaques at the plasma membrane, can be internalized into one of the two adjoining cells as unique double-membrane endocytic structures called annular gap junctions (AGJ) or connexosomes (Gumpert et al, 2008;Piehl et al, 2007;Gilleron et al, 2011;Falk et al, 2009). Following internalization from the plasma membrane, connexins are degraded by endolysosomal and autophagic mechanisms (Hesketh et al, 2010;Lichtenstein et al, 2011;Bejarano et al, 2012;Leithe et al, 2011;Falk et al, 2009;Laing et al, 1997;Leithe et al, 2006;Leithe et al, 2009;Qin et al, 2003;VanSlyke & Musil, 2005;Berthoud et al, 2000;Laird, 2006;Laird et al, 1995). These mechanisms along with proteasome-mediated ER-associated degradation regulate the rapid turnover and relatively short, 1.5 -5 hour half-life of connexins VanSlyke & Musil, 2002).…”