Defining a minimal motif required to prevent connexin oligomerization in the endoplasmic reticulum.
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.
Maza
Jose
J
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Das Sarma
Jayasri
J
Koval
Michael
M
D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D018031:Connexin 43 / Q000737:chemistry* / Q000378:metabolism; D017630:Connexins / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000494:pharmacology; D014162:Transfection; D000097002:Gap Junction beta-1 Protein
D018031:Connexin 43; D017630:Connexins; D017931:DNA Primers; D003902:Detergents; D008239:Lysine; D011993:Recombinant Fusion Proteins; D013395:Sucrose; D000097002:Gap Junction beta-1 Protein