Interaction of Atg8, Shp1, and Cdc48. The split-ubiquitin system proposed preferential interaction of Shp1 with lipidated Atg8. Protein interaction results in slower growth on medium lacking uracil. Dilutions were dropped on medium with (a) and without (b) uracil. Ste14-Cub/Nui-Ubc6, positive; Ste14-Cub/pRS314, negative control. (c) Shp1-HA from lysates (L) of starved atg4Δ cells expressing GFP-Atg8-FG were immunoprecipitated with HA antibodies. S, supernatant; IP, immunoprecipitate. Immunoblots with GFP (top) and HA antibodies (bottom) are shown. *, cross-reaction. (d) Atg8 N terminus aligned with mammalian homologues. (e) GST fusions on beads were incubated with lysates of cells chromosomally expressing Shp1-HA with native promoter. (f) As in panel e, GST fusions were incubated with lysate from cells chromosomally expressing Shp1-HA and Cdc48-GFP with native promoters. AL, alkaline lysis; W, wash, PD, bound. Immunoblots with HA (top) or GFP antibodies (bottom). The Cdc48-GFP double band might be a result of proteolysis.