Figure 4From: VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPaseVAPB interaction with FAF1 and p97 is stimulated upon proteasome inhibition. (A) Proteasome inhibition enhances ubiquitin, p97 and FAF1 binding to Flag-VAPB, while ASNA1 binding remains largely unchanged. U2OS cells expressing Flag-VAPB from a tetracycline-inducible promoter were grown in the presence of 100Â ng/ml tetracycline for 24Â hr or left untreated as a control. Flag-VAPB was immunoprecipitated using anti-Flag beads. (B) Proteasome inhibition enhances ubiquitin, p97 and FAF1 binding to endogenous VAPB, while ASNA1 binding was only slightly increased. Endogenous VAPB was immunoprecipitated from U2OS cells using anti-VAPB antibodies cross-linked to Protein A-beads. Uncoupled beads were used as control. (C, D) The binding of ubiquitinated proteins to VAPB is largely mediated by FAF1. U2OS cells were treated with the indicated siRNA oligos. Flag-VAPB (C) or endogenous VAPB (D) were immunoprecipitated as described above. Depletion of FAF1 strongly reduces the binding of ubiquitinated proteins to Flag- or endogenous VAPB. (E) The K87D M89D double mutant (KM-DD) of VAPB is defective in p97, FAF1, ASNA1 and ubiquitin binding. Flag-VAPB full-length WT, KM-DD and truncated, C-terminal half (C-Ter) and N-terminal half (MSP), were immunoprecipitated from U2OS cells. The MSP domain of VAPB is sufficient to interact with p97, FAF1 and ASNA1. KM-DD as well as the truncation lacking the MSP domain (C-Ter) are defective in binding poly-ubiquitinated proteins and seem to interact preferentially with oligo-ubiquitinated proteins. (F) The binding of ubiquitinated proteins to VAPB is reduced in cells treated with ASNA1 siRNA. U2OS cells expressing Flag-VAPB from a tetracycline-inducible promoter were treated with the indicated siRNA oligos. Depletion of ASNA1 reduces ubiquitin and BAG6 binding, but not FAF1 binding, to Flag-VAPB. C-Ter, C-terminal half; IP, immunoprecipitate; KM-DD, K87D M89D double mutant; Luc, luciferase.Back to article page