Limits...
Connexin46 is retained as monomers in a trans-Golgi compartment of osteoblastic cells.

Koval M, Harley JE, Hick E, Steinberg TH - J. Cell Biol. (1997)

Bottom Line: In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers.These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell.Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA. koval@id.wustl.edu

ABSTRACT
Connexins are gap junction proteins that form aqueous channels to interconnect adjacent cells. Rat osteoblasts express connexin43 (Cx43), which forms functional gap junctions at the cell surface. We have found that ROS 17/2.8 osteosarcoma cells, UMR 106-01 osteosarcoma cells, and primary rat calvarial osteoblastic cells also express another gap junction protein, Cx46. Cx46 is a major component of plasma membrane gap junctions in lens. In contrast, Cx46 expressed by osteoblastic cells was predominantly localized to an intracellular perinuclear compartment, which appeared to be an aspect of the TGN as determined by immunofluorescence colocalization. Hela cells transfected with rat Cx46 cDNA (Hela/Cx46) assembled Cx46 into functional gap junction channels at the cell surface. Both rat lens and Hela/Cx46 cells expressed 53-kD (nonphosphorylated) and 68-kD (phosphorylated) forms of Cx46; however, only the 53-kD form was produced by osteoblasts. To examine connexin assembly, monomers were resolved from oligomers by sucrose gradient velocity sedimentation analysis of 1% Triton X-100-solubilized extracts. While Cx43 was assembled into multimeric complexes, ROS cells contained only the monomer form of Cx46. In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers. These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell. Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.

Show MeSH

Related in: MedlinePlus

Sucrose gradient analysis of Cx43 and Cx46 oligomerization. ROS cells were harvested and homogenized, and Triton  X-100 was added to a final concentration of 1%. After an incubation for 30 min at 4°C, the preparation was centrifuged at 50,000 g  for 30 min, and the resulting Triton-soluble fraction was layered  onto a 5–20% sucrose gradient containing 0.1% Triton X-100  (with a 25% sucrose cushion). The gradient was centrifuged for  16 h at 148,000 g. Fractions were collected from the bottom of the  tube, diluted 1:1 with sample buffer, resolved by SDS-PAGE,  and electrophoretically transferred to PVDF membranes. Cx43 (a  and c) and Cx46 (b and d) were detected by immunoblotting (c  and d) and quantified by densitometry (a and b). Lanes in c and d  are labeled with the corresponding sample sucrose concentration,  which was determined by refractometry.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139843&req=5

Figure 9: Sucrose gradient analysis of Cx43 and Cx46 oligomerization. ROS cells were harvested and homogenized, and Triton X-100 was added to a final concentration of 1%. After an incubation for 30 min at 4°C, the preparation was centrifuged at 50,000 g for 30 min, and the resulting Triton-soluble fraction was layered onto a 5–20% sucrose gradient containing 0.1% Triton X-100 (with a 25% sucrose cushion). The gradient was centrifuged for 16 h at 148,000 g. Fractions were collected from the bottom of the tube, diluted 1:1 with sample buffer, resolved by SDS-PAGE, and electrophoretically transferred to PVDF membranes. Cx43 (a and c) and Cx46 (b and d) were detected by immunoblotting (c and d) and quantified by densitometry (a and b). Lanes in c and d are labeled with the corresponding sample sucrose concentration, which was determined by refractometry.

Mentions: Detergent-extracted connexin preparations were resolved into monomers and multimers by sucrose gradient velocity sedimentation (Fig. 9). Cx43 from ROS cells resolved into two fractions. The major fraction centered at 8% sucrose corresponds to Cx43 monomers, while the peak at 15% sucrose corresponds to multimers. Multimeric Cx43 consisted of 21.4 ± 2.8% (n = 3) of the total Triton X-100–soluble Cx43. The preponderance of Cx43 monomers over hexamers was expected, since the Triton-soluble fraction of Cx43 is likely to contain mostly newly synthesized Cx43 (39, 40). However, some monomeric Cx43 may be due to spontaneous disassembly of hexameric Cx43 during the course of the experiment.


Connexin46 is retained as monomers in a trans-Golgi compartment of osteoblastic cells.

Koval M, Harley JE, Hick E, Steinberg TH - J. Cell Biol. (1997)

Sucrose gradient analysis of Cx43 and Cx46 oligomerization. ROS cells were harvested and homogenized, and Triton  X-100 was added to a final concentration of 1%. After an incubation for 30 min at 4°C, the preparation was centrifuged at 50,000 g  for 30 min, and the resulting Triton-soluble fraction was layered  onto a 5–20% sucrose gradient containing 0.1% Triton X-100  (with a 25% sucrose cushion). The gradient was centrifuged for  16 h at 148,000 g. Fractions were collected from the bottom of the  tube, diluted 1:1 with sample buffer, resolved by SDS-PAGE,  and electrophoretically transferred to PVDF membranes. Cx43 (a  and c) and Cx46 (b and d) were detected by immunoblotting (c  and d) and quantified by densitometry (a and b). Lanes in c and d  are labeled with the corresponding sample sucrose concentration,  which was determined by refractometry.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139843&req=5

Figure 9: Sucrose gradient analysis of Cx43 and Cx46 oligomerization. ROS cells were harvested and homogenized, and Triton X-100 was added to a final concentration of 1%. After an incubation for 30 min at 4°C, the preparation was centrifuged at 50,000 g for 30 min, and the resulting Triton-soluble fraction was layered onto a 5–20% sucrose gradient containing 0.1% Triton X-100 (with a 25% sucrose cushion). The gradient was centrifuged for 16 h at 148,000 g. Fractions were collected from the bottom of the tube, diluted 1:1 with sample buffer, resolved by SDS-PAGE, and electrophoretically transferred to PVDF membranes. Cx43 (a and c) and Cx46 (b and d) were detected by immunoblotting (c and d) and quantified by densitometry (a and b). Lanes in c and d are labeled with the corresponding sample sucrose concentration, which was determined by refractometry.
Mentions: Detergent-extracted connexin preparations were resolved into monomers and multimers by sucrose gradient velocity sedimentation (Fig. 9). Cx43 from ROS cells resolved into two fractions. The major fraction centered at 8% sucrose corresponds to Cx43 monomers, while the peak at 15% sucrose corresponds to multimers. Multimeric Cx43 consisted of 21.4 ± 2.8% (n = 3) of the total Triton X-100–soluble Cx43. The preponderance of Cx43 monomers over hexamers was expected, since the Triton-soluble fraction of Cx43 is likely to contain mostly newly synthesized Cx43 (39, 40). However, some monomeric Cx43 may be due to spontaneous disassembly of hexameric Cx43 during the course of the experiment.

Bottom Line: In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers.These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell.Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA. koval@id.wustl.edu

ABSTRACT
Connexins are gap junction proteins that form aqueous channels to interconnect adjacent cells. Rat osteoblasts express connexin43 (Cx43), which forms functional gap junctions at the cell surface. We have found that ROS 17/2.8 osteosarcoma cells, UMR 106-01 osteosarcoma cells, and primary rat calvarial osteoblastic cells also express another gap junction protein, Cx46. Cx46 is a major component of plasma membrane gap junctions in lens. In contrast, Cx46 expressed by osteoblastic cells was predominantly localized to an intracellular perinuclear compartment, which appeared to be an aspect of the TGN as determined by immunofluorescence colocalization. Hela cells transfected with rat Cx46 cDNA (Hela/Cx46) assembled Cx46 into functional gap junction channels at the cell surface. Both rat lens and Hela/Cx46 cells expressed 53-kD (nonphosphorylated) and 68-kD (phosphorylated) forms of Cx46; however, only the 53-kD form was produced by osteoblasts. To examine connexin assembly, monomers were resolved from oligomers by sucrose gradient velocity sedimentation analysis of 1% Triton X-100-solubilized extracts. While Cx43 was assembled into multimeric complexes, ROS cells contained only the monomer form of Cx46. In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers. These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell. Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.

Show MeSH
Related in: MedlinePlus