Abstract
Connexin43 (Cx43) is a transmembrane protein that forms gap junction channels. Regulation of Cx43 turnover is one mechanism to control the level of intercellular communication that occurs through gap junction channels. Proteasomal degradation of Cx43 is regulated in part through CIP75, a ubiquitin-like and ubiquitin-associated domain containing protein. CIP75 interacts with endoplasmic reticulum-localized Cx43, as demonstrated through co-immunoprecipitation and immunofluorescence microscopy experiments. CIP75 also binds to free monoubiquitin and lysine 48-linked tetraubiquitin chains in vitro and binds to ubiquitinated proteins in cellular lysates. However, analysis of Cx43 that immunoprecipitated with CIP75 demonstrated that the Cx43 associated with CIP75 was not ubiquitinated, and a mutant form of Cx43 that lacked lysines capable of ubiquitination retained the capacity to interact with CIP75. These results suggest that although CIP75 can interact with ubiquitinated cellular proteins, its interaction with Cx43 and stimulation of Cx43 proteasomal degradation does not require the ubiquitination of Cx43.
Highlights
Gap junctions are plasma membrane channels that mediate direct cell to cell communication
We found that CIP75 interacted with ER-localized Cx43 through biochemical assays and immunofluorescence microscopy studies
The lack of ubiquitination of the Cx43 lysine to arginine mutants did not affect their ability to interact with CIP75 in the biochemical co-immunoprecipitation and immunofluorescence microscopy experiments
Summary
Cx43K9,13R Cx43K23R Cx43K69R Cx43K103–110R Cx43K115R Cx43K129–137R Cx43K145,147R Cx43K163R Cx43K189R Cx43K207R Cx43K235–242R Cx43K259,265R Cx43K265R Cx43K288R Cx43K303R Cx43K346,347R gcc ttg ggg agg ctt ctg gac agg gtc caa gcc gct gga ggg agg gtg tgg ctg tca gtg c acg tct gct atg aca ggt cct tcc cc gtg atg agg agg gaa gag agg cta aac agg aga gaa gag gag c gag gag ctc aga gtg gcc cag act gac g tgc acc tga ggc aga ttg aaa tca gga ggt tca ggt acg gga ttg gag cac ggc agg gtg aga atg agg ggc g gtt cta cac ctg cag gag aga tcc c gca tcc tct tca ggt ctg tct tcg agg ccc acg gag aga acc atc ttc atc atc ttc atg c cgt ctt ctt cag agg cgt tag gga tcg cgt gag ggg aag aag c gag ccc atc aag aga ctg cgg atc tcc aag ata cgc c gga tct cca aga tac gcc tac ttc aat ggc ctc ctc ctg ggt aca ggc tgg tta ctg g gcc gca att aca aca ggc aag cta gcg agc gac aac cag aat gcc aga aga gtt gct gct gg proteasome to be degraded. Because the regulation of connexin levels has a direct correlation with gap junctional intercellular communication, understanding the mechanism by which connexin levels are regulated is critical. In this present work, we have found that CIP75 was a ubiquitin-binding protein that interacts with Cx43 located in the ER and that this interaction increases upon inhibition of proteasomal degradation. In contrast with the more typical ubiquitin-dependent pathway for proteasomal degradation, the CIP75-Cx43 interaction was not dependent on Cx43 ubiquitination These results suggest that CIP75 regulates proteasomal degradation of non-ubiquitinated Cx43, perhaps in concert with mediating the degradation of other ubiquitinated cellular protein substrates
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