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Originally published as MBoC in Press, 10.1091/mbc.E09-01-0062 on March 18, 2009

Vol. 20, Issue 9, 2451-2463, May 1, 2009

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Conformational Maturation and Post-ER Multisubunit Assembly of Gap Junction Proteins

Judy K. VanSlyke*, Christian C. Naus{dagger}, and Linda S. Musil*

*Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239; and {dagger}Department of Cellular and Physiological Sciences, Life Sciences Institute, The University of British Columbia, Vancouver, BC V6T 1Z3

Submitted January 21, 2009; Accepted March 6, 2009
Monitoring Editor: Jeffrey L. Brodsky

For all previously well-characterized oligomeric integral membrane proteins, folding, multisubunit assembly, and recognition of conformationally immature molecules for degradation occurs at their organelle of synthesis. This cannot, however, be the case for the gap junction–forming protein connexin43 (Cx43), which when endogenously expressed undergoes multisubunit assembly into connexons only after its transport to the trans-Golgi network. We have developed two novel assays to assess Cx43 folding and assembly: acquisition of resistance of disulfide bonds to reduction by extracellularly added DTT and Triton X-114 detergent phase partitioning. We show that Cx43 synthesized at physiologically relevant levels undergoes a multistep conformational maturation process in which folding of connexin monomers within the ER is a prerequisite for multisubunit assembly in the TGN. Similar results were obtained with Cx32, disproving the widely reported contention that the site of endogenous β connexin assembly is the ER. Exogenous overexpression of Cx43, Cx32, or Cx26 allows these events to take place within the ER, the first example of the TGN and ER as alternative sites for oligomeric assembly. Our findings also constitute the first biochemical evidence that defective connexin folding is a cause of the human disorder X-linked Charcot-Marie-Tooth disease.

This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E09-01-0062) on March 18, 2009.

Address correspondence to: Linda S. Musil (musill{at}OHSU.edu)

Abbreviations used: BFA, brefeldin A; CCCP, carbonyl cyanide m-chloro-phenyl hydrazone; CHX, cycloheximide; CMTX, X-linked Charcot-Marie-Tooth disease; Cx26, Cx32, Cx43, connexin26, -32, and 43, respectively; DTT, dithiothreitol; N-LS, N-lauroyl sarcosine; TX-100 and -114, Triton X-100 and -114, respectively




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