Biochemical Analysis of Connexin43 Intracellular Transport, Phosphorylation, and Assembly into Gap Junctional Plaques

• Published in: The Journal of cell biology Vol. 115; no. 5; pp. 1357 - 1374
• Main Authors: Musil, Linda S., Goodenough, Daniel A.
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01.12.1991; The Rockefeller University Press
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Biological Transport; Biotin; Cell communication; Cell culture techniques; Cell Line; Cell lines; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cell membranes; Cells; Cellular metabolism; connexin-43; Connexins; Detergents; Epithelial cells; Fundamental and applied biological sciences. Psychology; Gap junctions; Membrane Proteins - metabolism; Membrane Proteins - ultrastructure; Microscopy, Electron; Octoxynol; Phosphorylation; Polyethylene Glycols; Proteins; Solubility; Proteins; Intracellular transport; Vertebrata; Phosphorylation; Cell line; Mammalia; Molecular association; Cell junction
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.115.5.1357

We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mrspecies (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2(Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20°C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2at 37°C. Connexin43-NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2form.