FIGURE 1.

The amino acid sequence PVEKAPR is sufficient for Rsp5 interaction. A, scheme representation of the cytoplasmic N-terminal sequences of the indicated proteins, which were expressed as C-terminal GST fusions to mimic the presentation of these proteins at the membrane. The Cps1 acceptor lysine is highlighted in red. B, GST constructs described in A were bound to glutathione-Sepharose beads and incubated with purified His-MBP-Rsp5. Bound material was detected with anti-MBP antibody (Rsp5) or Coomassie Brilliant Blue staining (GST baits). C, live cell imaging of the indicated GFP-tagged cargo was examined in wild type or rsp5Δ yeast co-expressing either DsRed-Rsp5^WT or a hypoactive HECT domain mutant (G753I) as the only source of Rsp5. Scale bar represents 5 microns. D, indicated GST fusions were bound to glutathione-Sepharose beads and incubated with the ubiquitination reaction components (E1, E2, MgATP, ubiquitin, biotin-ubiquitin) and His-MBP-tagged Rsp5. Ubiquitin-modified species were visualized by avidin-HRP to evaluate the ability of Rsp5 to modify these peptides in vitro.