Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation.
Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses. We report here that chick connexin43 is remarkably similar to its rat counterpart in primary amino acid sequence and in several key structural features as deduced by molecular cDNA cloning. The cross-reactivity of an anti-rat connexin43 serum with chick connexin43 permitted definitive immunocytochemical localization of chick connexin43 to lens epithelial gap junctional plaques and examination of the biosynthesis of connexin43 by metabolic radiolabeling and immunoprecipitation. We show that chick lens cells synthesize connexin43 as a single, 42-kD species that is efficiently posttranslationally converted to a 45-kD form. Metabolic labeling of connexin43 with 32P-orthophosphate combined with dephosphorylation experiments reveals that this shift in apparent molecular weight is due solely to phosphorylation. These results indicate that embryonic chick lens is an appropriate system for the study of connexin43 biosynthesis and demonstrate for the first time that connexin43 is a phosphoprotein.
Musil
L S
LS
Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, Massachusetts 02115.
Beyer
E C
EC
Goodenough
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D002642:Chick Embryo; D003001:Cloning, Molecular; D017630:Connexins; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D007908:Lens, Crystalline / Q000032:analysis / Q000196:embryology / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D051381:Rats
D017630:Connexins; D008565:Membrane Proteins; D004247:DNA