SBDS interacts directly with the GTPase EFL1 to jointly trigger eIF6 eviction. (A) In vivo interaction between endogenous SBDS and EFL1^FLAG. Lyzates from eflA^FLAG cells treated with DSP (+) or vehicle (DMSO) alone (-DSP) were immunoprecipitated with isotype control (IgG), anti-SBDS or anti-FLAG antibodies and the indicated proteins visualized by immunoblotting. Input represents 5% material used for immunoprecipitations. (B) In vivo interaction between endogenous SBDS and EFL1^FLAG is RNA-dependent. Lyzates were immunoprecipitated as in panel A after treatment with ribonuclease (RNase) before (bottom) or after (top) incubation with DSP. (C) Direct interaction between SBDS and EFL1^FLAG in vivo. Lyzates from eflA^FLAG cells treated with the crosslinker DSS were immunoprecipitated as in panel A. Native (arrowheads) and crosslinked (brackets) SBDS and EFL1^FLAG were visualized by immunoblotting (IB). Cell lysate untreated with DSS (1% of input) was loaded as a control (left). *Indicates mouse IgG heavy chain. (D) Human SBDS and EFL1 jointly catalyze eIF6 release. The indicated combinations of recombinant human SBDS and EFL1 (wild-type or the catalytically inactive mutants EFL1^T33A and EFL1^H96A) were incubated with pre-60S subunits purified from sbdS^int(ts) cells in the presence of GTP, GDP or the nonhydrolysable analog GDPNP. “Free” and “bound” fractions from sucrose cushions were immunoblotted to detect eIF6 and Rpl8.