Abstract
In contrast to most multimeric transmembrane complexes that oligomerize in the endoplasmic reticulum (ER), the gap junction protein connexin43 (Cx43) oligomerizes in an aspect of the Golgi apparatus. The mechanisms that prevent oligomerization of Cx43 and related connexins in the ER are not well understood. Also, some studies suggest that connexins can oligomerize in the ER. We used connexin constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, Cx43-HKKSL was retained in the ER and prevented from oligomerization. However, another ER-retained HKKSL-tagged connexin, Cx32-HKKSL, had the capacity to oligomerize. Because this suggested that Cx43 contains a motif that prevented oligomerization in the ER, a series of HKKSL-tagged and untagged Cx32/Cx43 chimeras was screened to define this motif. The minimal motif, which prevented ER oligomerization, consisted of the complete third transmembrane domain and the second extracellular loop from Cx43 on a Cx32 backbone. We propose that charged residues present in Cx43 and related connexins help prevent ER oligomerization by stabilizing the third transmembrane domain in the membrane bilayer.
Highlights
As an alternate approach, we have used connexins containing a dilysine endoplasmic reticulum (ER) retention/retrieval motif, HKKSL, to study early events in oligomerization of Cx32 and Cx43 [19]
We propose that charged residues present in Cx43 and related connexins help prevent ER oligomerization by stabilizing the third transmembrane domain in the membrane bilayer
Connexin mutations associated with human disease frequently interfere with proper connexin trafficking and assembly, causing mutant connexins to improperly accumulate in intracellular compartments such as the endoplasmic reticulum (ER)1 or aspects of the Golgi apparatus (8 –10)
Summary
We have used connexins containing a dilysine ER retention/retrieval motif, HKKSL, to study early events in oligomerization of Cx32 and Cx43 [19]. We found that ER-retained Cx32HKKSL had the capacity to oligomerize, whereas Cx43HKKSL did not [19]. This suggests that Cx43-HKKSL contains a motif required to inhibit oligomerization in the ER. Gap junction channels are formed by a family of proteins known as connexins [1,2,3,4,5,6,7]. A complete gap junction channel is formed when a connexin hexamer in the plasma membrane of one cell binds to a hexamer in an adjacent cell. Formation of connexin hexamers (oligomerization) occurs in intracellular compartments prior to transport to the plasma membrane. § To whom correspondence should be addressed: Emory University School of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, Whitehead Biomedical Research Bldg., 615 Michael St., Suite 205M, Atlanta, GA 30322
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