Targeted gap junction protein constructs reveal connexin-specific differences in oligomerization.
To define further the mechanisms of gap junction protein (connexin (Cx)) oligomerization without pharmacologic disruption, we have examined the transport and assembly of connexin constructs containing C-terminal di-lysine-based endoplasmic reticulum (ER) (HKKSL) or ER-Golgi intermediate compartment (AKKFF) targeting sequences. By immunofluorescence microscopy, Cx43-HKKSL transiently transfected into HeLa cells showed a predominantly ER localization, although Cx43-AKKFF was localized to the perinuclear region of the cell. Sucrose gradient analysis of Triton X-100-solubilized connexins showed that either Cx43-HKKSL or Cx43-AKKFF expressed alone by HeLa cells was maintained as an apparent monomer. In contrast to Cx43-HKKSL, Cx32-HKKSL was maintained in the ER as stable hexamers, consistent with the notion that Cx32 and Cx43 oligomerization occur in distinct intracellular compartments. Furthermore, Cx43-HKKSL and Cx43-AKKFF inhibited trafficking of Cx43 and Cx46 to the plasma membrane. The inhibitory effect was because of the formation of mixed oligomers between Cx43-HKKSL or Cx43-AKKF and wild type Cx43 or Cx46. Taken together, these results suggest that Cx43-HKKSL and Cx43-AKKFF recirculate through compartments where oligomerization occurs and may be maintained as apparent monomers by a putative Cx43-specific quality control mechanism.
Das Sarma
Jayasri
J
University of Pennsylvania School of Medicine, Department of Physiology and Institute for Environmental Medicine, Philadelphia, Pennsylvania 19104, USA.
Wang
Fushan
F
Koval
Michael
M
D001704:Biopolymers; D002450:Cell Communication; D017630:Connexins / Q000737:chemistry / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D013347:Subcellular Fractions / Q000378:metabolism
D001704:Biopolymers; D017630:Connexins; D004151:Dipeptides; C024575:lysyllysine