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Figure 1 Vps9 binds to monoubiquitin. (A) Lysate prepared from cells expressing Vps9-HA was incubated with Sepharose beads bound to GST, GST–Ub, monoubiquitin (Ub–Seph) or no protein (Seph). Total lysate (10% volume) and proteins eluted from each type of bead were analyzed by SDS–PAGE, followed by an anti-HA immunoblot. (B) Lysate from E.coli expressing His6-Vps9 was incubated with Sepharose or Ub–Sepharose beads. Total lysate and eluted proteins were separated on a 16.5% Tris-tricine gel and analyzed by Coomassie Blue staining or by immunoblotting with anti-histidine antiserum.
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 | Figure 2 The CUE domain of Vps9 interacts with ubiquitin directly. (A) Schematic of Vps9 indicating defined domains (http://smart.embl-heidelberg.de). Fragments of Vps9 fused to the Gal4 activation domain (AD) were assayed for interaction with UbK48R fused to the Gal4-binding domain (BD) by the yeast two-hybrid method. Growth on medium lacking histidine or adenine indicated a positive interaction. Growth on medium lacking adenine indicated a stronger interaction than growth on medium lacking histidine. The interaction between UbK48R and individual domains was quantified by assaying  -galactosidase activity in cell lysates. The background resulting from a strain co-expressing BD alone and AD alone was normalized to 1. (B) Bacterial lysates from cells expressing C-terminal fragments of Vps9 were incubated with Sepharose or Ub–Sepharose. Total lysates and eluted proteins were analyzed by Coomassie Blue staining. The arrowheads indicate the mobilities of Vps9 fragments. An endogenous bacterial polypeptide (*) also bound to Ub–Sepharose.
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Figure 3 Conserved CUE domain residues are important for monoubiquitin binding. Equal amounts of His6-tagged Vps9 CUE domain (408–451) and the indicated mutant variants were immobilized on metal affinity beads and incubated with bacterial lysates expressing GST or GST–Ub. After extensive washing, the beads were boiled. Lysates and eluted proteins were separated on a 15% SDS– polyacrylamide gel and analyzed by Coomassie Blue staining.
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 | Figure 4 The CUE motif is a general ubiquitin-binding domain. (A) CUE domains from yeast CUE proteins and a control SH3 domain from the yeast Rvs167 protein were expressed as His6-tagged polypeptides in E.coli. Binding to GST–Ub was performed as described in the legend to Figure 3, except GST/GST–Ub total lysates and eluted proteins were separated on a 16.5% Tris-tricine gel. Vps9 (amino acids 408–451), Cue1 (amino acids 65–106), Cue2-1 (amino acids 8–50), Cue2-2 (amino acids 55–97), Cue3 (amino acids 316–358), Cue4 (amino acids 74–115), Cue5 (amino acids 97–139) and SH3 (Rvs167 amino acids 428–482). (B) ITC analysis of Vps9-CUE and Cue1-CUE binding to ubiquitin. Titration curves are shown for the Vps9 and Cue1 CUE domains. Inset: a representative experimental ITC trace of Vps9-CUE. The differential heat signals from injections of 4 mM ubiquitin into 200  M Vps9 or Cue1 CUE domains are shown (after subtraction of blank data as described in Materials and methods). (C) CUE domains from yeast and human CUE proteins were analyzed as in (A). Tollip CUE (amino acids 229–271). (D) Binding of ubiquitin chains to Vps9 and Cue1 CUE domains. Oligo- and polyubiquitin chains (Ub2 and greater) were incubated with Vps9 and Cue1 CUE domains immobilized on metal affinity beads. Total ubiquitin chains (total) and ubiquitin in the flow-through (FT) and bound (B) fractions were fractionated by SDS–PAGE and detected on an immunoblot with ubiquitin antiserum.
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Figure 5 A CUE domain FP motif is important for binding to monoubiquitin. (A) Alignment of yeast CUE domains identified by database searches for sequences similar to regions of Cue1 and Tollip (Ponting, 2000). The CUE domain invariant proline and highly conserved di-leucine motif are highlighted in dark gray. X-Phe residues that precede the invariant proline are highlighted light gray. (B) Equal amounts of His6-tagged Vps9 CUE domain (408–451) and the indicated mutant variants were immobilized on metal affinity beads. Binding to GST and GST–Ub was performed as described in the legend to Figure 3. (C) Equal amounts of His6-tagged Cue1 CUE domain (amino acids 65–106) and the indicated mutant variants were immobilized on metal affinity beads. Binding to GST and GST–Ub was performed as described in the legend to Figure 3, except that total lysates and eluted proteins were analyzed by an anti-GST immunoblot and by Coomassie Blue staining.
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 | Figure 6 CUE domain binding requires Ile44 of ubiquitin. The Vps9 CUE domain (residues 408–451) was immobilized on metal affinity beads and incubated with bacterial lysates expressing GST, GST–Ub, GST–UbI44A or GST–UbF4A. The lysates and eluted proteins were analyzed by Coomassie Blue staining. The mobilities of the CUE domain, GST and GST–Ub are indicated.
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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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 | Figure 8 Ubiquitin-binding CUE proteins. Proteins that carry a CUE domain characterized by an FP and a di-leucine-like sequence, and therefore are likely to bind ubiquitin efficiently, are shown, and defined domains are indicated (http://smart.embl-heidelberg.de). Cylinders indicate the predicted transmembrane domain.
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