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

doi:10.1074/jbc.M002608200

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

From the Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, Oregon 97201

Abstract

Connexins, the integral membrane protein constituents of gap junctions, are degraded at a rate (t = 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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Vollum Institute for Advanced Biomedical Research L474, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Rd., Portland, OR 97201. Tel.: 503-494-1300; Fax: 503-494-8230; E-mail: Musill@ohsu.edu.
Published, JBC Papers in Press, June 2, 2000, DOI 10.1074/jbc.M002608200
Abbreviations:

Cx43

connexin43

Cx32

connexin32

NRK

normal rat kidney

ssS180L

serum-starved S180L

ZL₃VS

carboxybenzyl-leucyl-leucyl-leucine vinylsulfone

ALLN

N-acetyl-leu-leu-norleucinal

DTT

dithiothreitol

LY

Lucifer Yellow

CLQ

chloroquine

CHX

cycloheximide

CHO

Chinese hamster ovary

DMEM

Dulbecco's modified Eagle's medium

FCS

fetal calf serum

PBS

phosphate-buffered saline

HB

Hanks' balanced salt solution containing 1% bovine serum albumin
- Received March 27, 2000.
- Revision received May 10, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.