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. 2000 Jul;11(7):2315–2325. doi: 10.1091/mbc.11.7.2315

Figure 5.

Figure 5

The Apc11p RING-H2 finger mediates E1- and E2-dependent ubiquitination. (A) Wild-type (WT) or mutant (41, 44, 52, 62, 80, 81, and 91, as described in Figure 2) versions of Apc11p were tested for the ability to mediate polyubiquitination in vitro. Equivalent amounts of purified GST-Apc11p fusion proteins were incubated with purified E1, Ubc4 (E2), and GST-ubiquitin in reaction buffer containing ATP. After 90 min, reactions were stopped with 2x SDS gel loading buffer, separated by SDS-PAGE, and analyzed by immunoblotting with anti-GST monoclonal antibody. Lane 1 shows a reaction with GST-Apc11p which was stopped with SDS buffer before the addition of E1, E2, GST-ubiquitin, and reaction buffer (UN, unreacted). GST-ubiquitin-GST-Apc11p conjugates are indicated at right. Molecular weight markers, in kiloDaltons, are indicated at left. (B) Ubiquitination reactions were carried out with GST-Apc11p as described in A, using ubiquitin in place of GST-ubiquitin. Lanes 1 and 2 show reactions carried out in the absence of E1 or E2, respectively. Lane 3 shows a reaction carried out with the full complement (+) of components. GST-Apc11p and its ubiquitin-conjugated derivatives were detected by immunoblotting with anti-GST monoclonal antibody. Ubiquitin-GST-Apc11p conjugates are indicated at left. (C) Reactions were carried out as in A, with GST-Apc11p fusion proteins incubated in the presence of GST-ubiquitin, E1, and equivalent concentrations of either Ubc4p or Cdc34p. GST-fusion proteins were detected by immunoblotting with anti-GST monoclonal antibody. Autoubiquitinated Cdc34p is indicated by an arrow. (D) Ubiquitination reactions were carried out by incubating GST-ubiquitin and His-tagged Clb2p-HA or Clb2p lacking the destruction box (Clb2pΔDB-HA) in the presence of either His-tagged Apc11p or the c-Cbl RING finger purified as a GST-fusion protein. Reactions contained either Ubc4p or Cdc34p as indicated. Clb2 proteins and their ubiquitin-conjugated derivatives were detected by immunoblotting with anti-HA monoclonal antibody. GST-ubiquitin-Clb2p-HA conjugates are indicated at left.