The binding of BAG6 deletion mutants to an immobilized TA protein.

Radiolabelled full-length BAG6 (A) and fragments lacking the C-terminal 226 residues including the BAG domain (B), encoding the N-terminal 270 residues only (C) or lacking the N-terminal UBL (D) were synthesized in vitro as before and incubated with immobilized recombinant Sec61β with an intact (+TA) or deleted (−TA) tail-anchor region. Samples were processed as described for Figure 1 and bound material analyzed by SDS-PAGE and phosphorimaging. In this case BAG6 binding was sensitive to Triton-X100, and an arrow indicates the location of the relevant BAG6 derived product (cf. signals in lanes 3 and 6 in each panel). With full length BAG6, quantification showed that the signal obtained with the control protein lacking the TA region (Sec61β−TA) was less than 3% of that recovered using Sec61β with an intact tail-anchor (Sec61β+TA).