Regulation of Connexin Degradation as a Mechanism to Increase Gap Junction Assembly and Function

Linda S Musil,Anh-Chi N Le,Judy K Vanslyke,Lori M Roberts

doi:10.1074/jbc.275.33.25207

Linda S Musil, Anh-Chi N Le + Show 2 more

Open Access

https://doi.org/10.1074/jbc.275.33.25207

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Abstract

Connexins, the integral membrane protein constituents of gap junctions, are degraded at a rate (t(12) = 1.5-5 h) much faster than most other cell surface proteins. Although the turnover of connexins has been shown to be sensitive to inhibitors of either the lysosome or of the proteasome, how connexins are targeted for degradation and whether this process can be regulated to affect intercellular communication is unknown. We show here that reducing connexin degradation with inhibitors of the proteasome (but not with lysosomal blockers) is associated with a striking increase in gap junction assembly and intercellular dye transfer in cells inefficient in both processes under basal conditions. The effect of proteasome inhibitors on wild-type connexin stability, assembly, and function was mimicked by treatment of assembly-inefficient cells with inhibitors of protein synthesis such as cycloheximide. Sensitivity of connexin degradation to cycloheximide, but not to proteasome inhibitors, was abolished when connexins were rendered structurally abnormal by perturbation of essential disulfide bonds or by mutation. Our findings provide the first evidence that intercellular communication can be up-regulated at the level of connexin turnover and that a short-lived protein may be required for conformationally mature connexins to become substrates of proteasomal degradation.

Highlights

Gap junctions are ordered arrays of intercellular plasma membrane channels that directly link the cytosols of two adjoining cells
We have previously shown that each cell type assembles this Cx43 into connexons and transports them to the cell surface (30, 31)
In vivo as in tissue culture, cells range from assembly-efficient to assembly-incompetent (39) in their ability to oligomerize endogenously expressed, wild-type connexins into gap junctional plaques

Summary

Introduction

Gap junctions are ordered arrays of intercellular plasma membrane channels that directly link the cytosols of two adjoining cells. Pulse-chase analysis has demonstrated that connexin family members turn over with a half-life of only 1.5–5 h, even after incorporation into gap junctional plaques This rapid rate of degradation has been observed in a wide variety of mammalian systems including primary and established tissue culture cells (4, 5), whole organs (6), and intact animals (7). Experiments in which brefeldin A was used to block intracellular transport suggested that proteasome inhibitor-sensitive degradation of connexins occurs at the level of the ER (13) as well as after transport to the cell surface (12) Both sites have previously been implicated in proteasome-mediated degradation of other integral plasma membrane proteins (14). Highly unusual for an integral plasma membrane protein, rapid turnover kinetics are a common characteristic of mature, fully folded forms of cytoplasmic and nuclear proteins involved in signal transduction Before such proteins can be degraded by the proteasome, they must undergo a multistep. The mechanism is not yet clear, protein synthesis inhibitors slow the turnover and thereby prolong the activity of certain other (but not all) cytoplasmic proteasome substrates, including pp60v-src (20), estradiol-liganded estrogen receptors (21), and some forms of p53 (22)

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