Fig. 3

Connexin mutants are recognized by the ER quality control system. a Mass spectrometry volcano plot for FLAG-tagged Cx32^wt, immunoprecipitated in 1% digitonin from transfected HEK293T cells. Enriched proteins are denoted with their Uniprot gene name. Cx32 is shown in orange and ER chaperones investigated further in this study are highlighted in blue (EMC10) and green (Cnx). Either a rabbit monoclonal anti-FLAG antibody or a rabbit IgG isotype control was used. b Representative blots from immunoprecipitation experiments from HEK293T cells transfected with the indicated Cx32 constructs. Interaction of Cx32 with endogenous Cnx and EMC subunits was detected and increased for Cx32^L90H with both EMC4 and EMC10 (mean ± SEM, N ≥ 3, ns: nonsignificant, *P value < 0.05, two-tailed Student’s t tests). Quantifications were performed as described in the Methods section. c Transient knockdown of EMC5/10 by siRNA (average knockdown (KD) efficiencies are shown below the blots) increases glycosylation of a reporter site in loop 2 for Cx32^L90H. Monomeric species ±glycosylation are shown on the blot, indicative of the topologies depicted on the right. Changes in glycosylation, quantified as described in the Methods section, are shown on the right (mean ± SEM, N = 5, **P value < 0.01, two-tailed Student’s t tests). d Same as in b for co-transfected hamster BiP (mean ± SEM, N = 3, **P value < 0.01, two-tailed Student’s t tests). e Schematic of a reporter construct to assess BiP-TM region binding. The nonglycosylated immunoglobulin λ light chain C_L domain with its own ER import sequence is followed by a flexible linker connected to the TM sequence of interest shown below the schematic. TM segment 1 was inverted to allow for a type I topology. A C-terminal glycosylation site (NVT, marked in red) allows to assess membrane integration (no glycosylation) versus ER import (possible glycosylation). Membrane integration/ER import was assessed for Cx32 TM segment 1, 2, and 2 carrying the L90H mutation by transfection of the constructs into HEK293T cells and EndoH deglycosylation where indicated. f C_L-TM constructs were co-transfected with hamster BiP into HEK293T cells and their interaction was analyzed by co-immunoprecipitation experiments coupled to immunoblots