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The EMBO Journal (2003) 22, 1273–1281, doi:10.1093/emboj/cdg140 Figure 7 A ubiquitin-binding motif required for intramolecular monoubiquitylation, the CUE domain Susan C. Shih, Gali Prag, Smitha A. Francis, Myra A. Sutanto, James H. Hurley and Linda Hicke

Figure 7 CUE-dependent monoubiquitylation of Vps9 by Rsp5. (A) Plasmids encoding His6-tagged Vps9 and mutant variants were co-transformed into yeast cells with an empty vector, or with plasmids encoding wild-type (Ub) or c-myc-tagged ubiquitin (myc-Ub). Ubiquitin overexpression was induced in cells prior to preparing the yeast lysates. Cell lysates were prepared and analyzed by anti-His immunoblot. In cells that did not overexpress ubiquitin, a high molecular weight form of Vps9 that migrated at 77 kDa was observed (lane 1). The overexpression of wild-type ubiquitin yielded an increase in 77 kDa Vps9, as well as inducing the appearance of an uncharacterized Vps9 species (*). The overexpression of c-myc-ubiquitin resulted in increased mobility of the 77 kDa Vps9 species (compare lane 3 with lane 2). Deletion of the Vps9 CUE domain or introduction of the F420A mutation severely inhibited Vps9 ubiquitylation (lanes 5 and 7). (B) A centromere-based plasmid encoding HA-Vps9 was transformed into mdp1-1/rsp5 and isogenic wild-type cells (RSP5). Lysate from the multicopy VPS9 wild-type strain analyzed in (A) lane 1 was used to indicate the mobility of the Vps9 77 kDa species (RSP5, lane 8). Higher ubiquitylated forms of Vps9 are observed in one strain background (lane 9) in addition to monoubiquitylated Vps9. A lighter exposure in which the 77 kDa species is not visible in the wild-type strain lysate is shown to indicate that each strain contains equivalents amounts of non-ubiquitylated Vps9.

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