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. 2000 Nov 28;97(25):13871–13876. doi: 10.1073/pnas.250277297

Figure 4.

Figure 4

In vitro ubiquitination of VP40. VP40-WT and VP40-dPY were transcribed and translated in vitro (lanes 1 and 4, respectively). In vitro translated proteins were incubated with recombinant E1 and E2 (UBC8) proteins, ATP, ubiquitin, and either WT Rsp5 (lanes 2 and 5) or the C-A mutant form of Rsp5, as indicated (lanes 3 and 6). The position of the VP40 doublet (lanes 1–6) and multiubiquitinated forms of VP40 [VP40-ub(n); lane 2] are indicated. A positive control (52-kDa yeast protein encoded by the YHL002w gene; G.W. and J.H., unpublished data) for ubiquitination by Rsp5 is shown (lanes 7 and 8). The arrowhead depicts the location of the nonubiquitinated form of the 52-kDa protein. The presence or absence of WT Rsp5 or mut. Rsp5 is indicated by a + or −.