Figure 6.

High-level stable overexpression of Cx43 switches the site of onset of connexon assembly to the ER. (A) Stable C6 cell transfectants expressing Cx43 at levels threefold (Cx43-14) or 30-fold (Cx43-13) over that of untransfected parentals (C6) were labeled with [³⁵S]methionine for 4 h at 37°C, in either the absence (− BFA) or presence (+ BFA) of BFA. TX-100–soluble lysates were prepared and either cross-linked with EGS (+) or mock cross-linked (−) before immunoprecipitation of Cx43 and analysis by SDS-PAGE. (B–E) Characterization of connexon formation within the ER in Cx43-13 cells. (B) C6 Cx43-13 cells were pulse labeled for 15 min without (lanes 1 and 2) or with (lanes 3 and 4) a subsequent 2-h chase, all at 37°C and in the continuous presence of BFA. Cx43 was immunoprecipitated from Triton-soluble lysates either with (+) or without (−) prior cross-linking with EGS. (C) C6 Cx43-13 cells were labeled for 3.5 h at 37°C in the presence of BFA and lysed in TX-100, and the solubilized material was fractionated on 5–20% linear sucrose gradients. Fractions known to contain monomeric (5S; 8–11% sucrose) and connexon-assembled (9S; 14–17% sucrose) forms of Cx43 were collected and then subjected to cross-linking with 500 μg/ml DSP before immunoprecipitation of Cx43. (D) Cx43-13 cells labeled for 3 h at 37°C in the presence of BFA were lysed in 1% TX-114 at 4°C. After chemical cross-linking with EGS, one-half of the lysate was TX-114 phase-partitioned at 30°C into detergent-rich (D) and detergent-poor aqueous (A) phases, and the other half remained unpartitioned (T). Immunoprecipitated Cx43 was analyzed by SDS-PAGE. (E) Cx43-13 cells were pulsed for 1 h at 37°C in the presence of BFA either with (+ DTT) or without (− DTT) 2 mM DTT. Triton-soluble lysates were prepared, cross-linked with EGS (+) or left uncross-linked (−), and Cx43 immunoprecipitates were analyzed by SDS-PAGE.