Intralumenal docking of Cx36 channels in the ER isolates mis-trafficked protein

Abstract

The intracellular domains of connexins are essential for the assembly of gap junctions. For connexin 36 (Cx36), the major neuronal connexin, it has been shown that a dysfunctional PDZ binding motif interferes with electrical synapse formation. However, it is still unknown how this motif coordinates the transport of Cx36. In the present study, we characterize a phenotype of Cx36 mutants that lack a functional PDZ binding motif using HEK293T cells as an expression system. We provide evidence that an intact PDZ binding motif is critical for proper ER export of Cx36. Removing the PDZ binding motif of Cx36 results in ER retention and the formation of multi-membrane vesicles containing gap junction-like connexin aggregates. Using a combination of site directed mutagenesis and electron micrographs we reveal that these vesicles consist of Cx36 channels that docked prematurely in the ER. Our data suggest a model in which ER-retained Cx36 channels reshape the ER membrane into concentric whorls that are released into the cytoplasm.

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“…Correlation with the underlying ultrastructure rapidly allowed identification of these structures and suggested them to be originating from the endoplasmic reticulum ( Fig. 5 Bi, Ci), which is in line with recently published data (Tetenborg et al, 2022).…”

Trafficking of Connexin36 (Cx36) in the early secretory pathway

“…Cx36 and Cx36S318ter were previously used in Tetenborg et al, (19). C-terminal Cx36 mutants were generated using the Q5 site directed mutagenesis kit (E0554S; New England Biolabs, Ipswitch, MA) and corresponding primer pairs introducing gaps into the Cx36 coding sequence.…”

“…The Venus-PDZ1 (ZO-1) was generated by site directed mutagenesis of the Venus-ZO-1 expression vector (Addgene: 56394) using primer pairs that delete the entire ZO-1 coding sequence except for the first PDZ1 domain. V5-dGBP-TurboID was cloned into a mammalian expression vector and used previously (19, 21)…”

“…Yet, PBM-mediated interactions could regulate other steps of Cx36 hemichannel assembly earlier in the secretory pathway. Indeed, previous work from our group found that the C-terminal tip of Cx36 functions as an ER export signal, as Cx36 mutants that lacked the PBM displayed severe ER retention resulting in the formation of ER derived removal vesicles (19). In line with this observation, we identified perinuclear accumulations of Cx36-EGFP in AII amacrine cell somas of the Cx36-EGFP/Cx36 KO mouse strain, resembling the ER retention phenotype that was observed in transiently transfected cells (19).…”

“…Indeed, previous work from our group found that the C-terminal tip of Cx36 functions as an ER export signal, as Cx36 mutants that lacked the PBM displayed severe ER retention resulting in the formation of ER derived removal vesicles (19). In line with this observation, we identified perinuclear accumulations of Cx36-EGFP in AII amacrine cell somas of the Cx36-EGFP/Cx36 KO mouse strain, resembling the ER retention phenotype that was observed in transiently transfected cells (19). Thus, the loss of gap junction formation described for Cx36-EGFP/Cx36 KO mice (15, 16) may not be solely attributable to a lack of association with ZO-1 but could reflect a secretory deficit.…”