Figure 2.

Purified Bag6-Client Complexes Support Ubiquitination by the MLP Ligase

(A) Luciferase (Luc) and/or Bag6 was mixed as indicated and either left at 4°C or heated to 42°C for 20 min. After centrifugation, the soluble and insoluble fractions were analyzed by SDS-PAGE and Coomassie staining.

(B) Coomassie-stained gel showing Bag6-Luc or ΔUbl-Bag6-Luc complexes.

(C) Soluble luciferase (−), Bag6-Luc complex (B), or ΔUbl-Bag6-Luc complex (Δ) was added to reticulocyte lysate (RRL), Bag6-depleted lysate (ΔBag6-RRL), or buffer; supplemented with E1, E2, ATP, and Flag-ubiquitin; and incubated at 37°C for 30 min. The reaction was analyzed by anti-luciferase immunoblot either directly (Input) or after FLAG immunoprecipitation. The position of luciferase-ubiquitin conjugates is indicated. Asterisk denotes unmodified luciferase that nonspecifically binds the resin, particularly in the absence of lysate.

(D) Bag6-depleted lysate was fractionated over a 5%–25% sucrose gradient, and each fraction was tested for ubiquitination activity using Bag6-Luc complex as the substrate as in (C). The migration positions of hemoglobin (60 kD native size), Hsp90, Bag6 complex, and Cullin1 are indicated.

See also Figure S2.