Membrane integration of in vitro-translated gap junctional proteins

Co- and post-translational mechanisms

Jian-Ting Zhang, Mingang Chen, Cynthia I. Foote, Bruce J. Nicholson

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

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 or 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.

Original languageEnglish (US)
Pages (from-to)471-482
Number of pages12
JournalMolecular Biology of the Cell
Volume7
Issue number3
StatePublished - Mar 1996
Externally publishedYes

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Connexins
Membranes
Gap Junctions
Proteins
trans-Golgi Network
Microsomes
Detergents
Hepatocytes
Amino Acid Sequence
Membrane Proteins
Hydrolysis
Nucleotides
Complementary DNA
Cell Membrane
In Vitro Techniques

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Membrane integration of in vitro-translated gap junctional proteins : Co- and post-translational mechanisms. / Zhang, Jian-Ting; Chen, Mingang; Foote, Cynthia I.; Nicholson, Bruce J.

In: Molecular Biology of the Cell, Vol. 7, No. 3, 03.1996, p. 471-482.

Research output: Contribution to journalArticle

Zhang, Jian-Ting ; Chen, Mingang ; Foote, Cynthia I. ; Nicholson, Bruce J. / Membrane integration of in vitro-translated gap junctional proteins : Co- and post-translational mechanisms. In: Molecular Biology of the Cell. 1996 ; Vol. 7, No. 3. pp. 471-482.
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