CMT X: Conformational Maturation and Post-ER Multisubunit Assembly of Gap Juncti

[Guest guest]

Mol Biol Cell. 2009 Mar 18.

Conformational Maturation and Post-ER Multisubunit Assembly of Gap Junction

Proteins.

Vanslyke JK, Naus CC, Musil LS.

Department of Biochemistry and Molecular Biology, Oregon Health and Science

University, Portland, OR 97239; Department of Cellular and Physiological

Sciences, Life Sciences Institute, The University of British Columbia,

Vancouver, BC V6T 1Z3, Canada.

Monitoring Editor: L. Brodsky

For all previously well-characterized oligomeric integral membrane proteins,

folding, multisubunit assembly, and recognition of conformationally immature

molecules for degradation occur at the organelle in which they are synthesized.

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

multi-step 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 beta connexin assembly is the ER.

Exogenous overexpression of Cx43, 32, or 26 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.