Membrane integration of in vitro-translated gap junctional proteins: co- and post-translational mechanisms.

AUTOR(ES)

Zhang, J T

RESUMO

Connexins (Cx) are protein components of gap junction channels that permit the passage of small molecules between neighboring cells. cDNAs of a large family of connexins have been isolated and sequenced. A gap junction channel consists of two connexons, one from each cell in contact, composed of six connexin subunits. It has been suggested by Musil and coworkers that the oligomerization of formation of a connexon occurs at the level of the trans-Golgi network. In the present study, we initiated an analysis of the early stages of protein synthesis and membrane insertion of Cx32 and Cx26, two connexins that we have demonstrated are co-expressed in the same junctions in hepatocytes. Using an in vitro transcription and a coupled cell-free translation and translocation system, we observed that both Cx32 and Cx26 could insert into microsome membranes co-translationally, producing a topological structure indistinguishable from that in isolated gap junctions. To our surprise, Cx26 could also insert into membranes post-translationally with a native orientation. This post-translational membrane insertion process is dependent on nucleotides but not their hydrolysis. Cx32, on the other hand, could not insert into membranes post-translationally. These disparate properties of Cx32 and Cx26 are not due to the significant difference in the lengths of their C-terminal domains, but rather to their internal amino acid sequences. These observations raise the possibility that there may be another pathway for Cx26 to insert into membranes in cells and this feature may be important for the regulation of its functions. These findings may also lead us to a new approach to reconstitution without detergent extraction.

Documentos Relacionados

• Myristyl amino-terminal acylation of murine retrovirus proteins: An unusual post-translational protein modification
• Post-translational modification and processing of Escherichia coli prolipoprotein in vitro.
• Regulation of membrane IgM expression in secretory B cells: translational and post-translational events.
• Post-translational modification of neuronal proteins: evidence for transglutaminase activity in R2, the giant cholinergic neuron of Aplysia.
• Post-translational transport of proteins into microsomal membranes of Candida maltosa.