1. E3-Independent Monoubiquitination of Ubiquitin-Binding Proteins
Daniela Hoeller, Christina-Maria Hecker, Sebastian Wagner, Vladimir Rogov, Volker Dötsch, Ivan Dikic 
Molecular Cell 
Volume 26, Issue 6, Pages (June 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
UBD-Containing Proteins Are Ubiquitinated in an E3-Independent Manner In Vitro
Bacterially expressed and purified substrates were incubated with 200 nM E1, ATP, 4 mM Flag-Ub, and 1 μM of the indicated E2 enzyme. Samples were subjected to SDS-PAGE and analyzed by immunoblotting using anti-Flag antibody (upper panel) and Ponceau S staining (lower panel). GST-Stam2 (A). GST-Eps15 (B). GST-Pol ι C-terminal half (C). GST-Pol κ C-terminal half (D). GST-HDAC6 (E). His6-Sts1 (F).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
Isolated UBDs Are Sufficient to Promote E3-Independent Ubiquitination, which Requires an Intact Ub/UBD Interface
(A) UBDs fused to either GST or NusA were purified from bacteria and incubated with 200 nM E1, 4 mM Flag-Ub, ATP, and 1 μM of either UBCH5A or the whole panel of different E2s. Samples were subjected to SDS-PAGE and analyzed by immunoblotting using anti-Flag antibodies.
(B) Wild-type (WT) and point mutants of UBDs of GST-Pol ι and GST-HDAC6 and a deletion mutant of the UBDs in Eps15 were purified from bacteria. WT and mutants of HA-Stam2 and Flag-Sts1 were isolated from 293T cells. Purified proteins were incubated with E1, ATP, Flag-Ub, and with UbcH7 or UbcH5A. Samples were subjected to SDS-PAGE and analyzed by immunoblotting using a tag-specific antibody.
(C) Bacterially expressed and purified GST-Eps15 (C terminus), GST-Sts1-UBA, and GST-Pol ι were incubated with E1/ATP, UBCH5A, and Ub WT or Ub Ile44 mutant for 2 hr at 37°C. Samples were analyzed by SDS-PAGE and immunoblotting using anti ubiquitin-specific antibody (P4D1, Santa Cruz).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3 UBD-Containing Proteins Do Not Act In trans
(A) Sts2 WT is unable to monoubiquitinate Sts2ΔUBA.
(B) Hrs WT cannot promote monoubiquitination of Stam2-UIM∗. HEK 293T lysates expressing the indicated proteins were subjected to SDS-PAGE and immunoblotting with tag-specific antibodies.
(C) GST-Nedd4, but not GST-Pol ι or GST-Stam2, can promote polymerization of monomeric Ub in vitro. Bacterial lysates containing GST-Stam2, GST-Pol ι, and GST-Nedd4 were incubated with E1, ATP, E2 (UbcH5A), and purified Flag-Ub. Polymerized Ub was detected by SDS-PAGE and immunoblotting using an anti Flag antibody (P4D1).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4 E3-Independent Monoubiquitination Is Relevant In Vivo
(A) siRNA-mediated downregulation of Nedd4 and Aip4 does not affect monoubiquitination of Eps15 and Hrs. HeLa cells were first transfected with pTER-shNedd4/pTER-shAip4 or pTER-shControl. Twenty-four hours later, Flag-Hrs or Flag-Eps15 was contransfected with HA-Ub. Another 48 hr later, cells were lysed and analyzed by SDS-PAGE and immunoblotting. The antibody used for detection of endogenous Nedd4 crossreacts with Aip4.
(B) FRET signals obtained for Sts2ΔPGM/UbcH7 WT (1), Sts2ΔPGM/UbcH7-F63N (2), Sts2ΔPGM/UbcH7-C86A (3), and Sts2ΔPGMΔUBA/UbcH7 WT (4). Dimerization-deficient Cit-Sts2ΔPGM and CFP-Sts2ΔPGM were used as a negative control (5). FRET can be observed when the distance between the FRET partners is 1.6 × R0. For CFP- and Citrine-tagged proteins, R0 is 4.9 and the maximal distance to obtain a FRET signal is therefore 7.8 nm (Verveer et al., 2006). Data are expressed as means of FRET efficiencies with p < 0.05, n = 10). The third column in the table shows the ratios between the CFP/YFP intensities to prove that both chromophores were expressed with similar efficiency in the bleached cell.
(C) Loss of Sts2 monoubiquitination after UbcH5/7 knockdown can be rescued by expression of UbcH5B F62N (E3 binding-deficient mutant) or UbcH5B WT. (Left) Extent of monoubiquitination of overexpressed Flag-Sts2 in the presence of endogenous Ub was quantified from three independent experiments with ImageJ. Monoubiquitination of Flag-Sts2 in control cells was set to 100%. The data represent the mean ± SEM of three experiments. (Right) Western blot of one representative experiment. 293T cells were transfected with the indicated oligos and Flag-Sts2 as described in the Experimental Procedures. Cells were lysed and analyzed by immunoblotting using the indicated antibodies.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions