Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques

Linda Musil, Daniel A. Goodenough

Research output: Contribution to journalArticle

589 Citations (Scopus)

Abstract

We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphor-ylated to a 46,000-Mr species (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 P2 form 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 (Tritonsoluble) 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-P2 at 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-P2 form.

Original languageEnglish (US)
Pages (from-to)1357-1374
Number of pages18
JournalJournal of Cell Biology
Volume115
Issue number5
StatePublished - Dec 1991
Externally publishedYes

Fingerprint

Connexin 43
Phosphorylation
Gap Junctions
Octoxynol
Cell Membrane
Biotinylation
Connexins

ASJC Scopus subject areas

  • Cell Biology

Cite this

Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques. / Musil, Linda; Goodenough, Daniel A.

In: Journal of Cell Biology, Vol. 115, No. 5, 12.1991, p. 1357-1374.

Research output: Contribution to journalArticle

@article{b72652bcfd04442681e2dcad574a9e3c,
title = "Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques",
abstract = "We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphor-ylated to a 46,000-Mr species (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 P2 form 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 (Tritonsoluble) 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-P2 at 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-P2 form.",
author = "Linda Musil and Goodenough, {Daniel A.}",
year = "1991",
month = "12",
language = "English (US)",
volume = "115",
pages = "1357--1374",
journal = "Journal of Cell Biology",
issn = "0021-9525",
publisher = "Rockefeller University Press",
number = "5",

}

TY - JOUR

T1 - Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques

AU - Musil, Linda

AU - Goodenough, Daniel A.

PY - 1991/12

Y1 - 1991/12

N2 - We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphor-ylated to a 46,000-Mr species (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 P2 form 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 (Tritonsoluble) 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-P2 at 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-P2 form.

AB - We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphor-ylated to a 46,000-Mr species (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 P2 form 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 (Tritonsoluble) 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-P2 at 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-P2 form.

UR - http://www.scopus.com/inward/record.url?scp=0025789648&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025789648&partnerID=8YFLogxK

M3 - Article

C2 - 1659577

AN - SCOPUS:0025789648

VL - 115

SP - 1357

EP - 1374

JO - Journal of Cell Biology

JF - Journal of Cell Biology

SN - 0021-9525

IS - 5

ER -