Fig. 6.

SCF-Fbxo28 selectively ubiquitylates Alcat1 at residue K183 and docks within the protein. (A) Schematic presentation of Alcat1 truncation mutants, which were tagged with V5. Amino acid positions are numbered. (B) The wild type (WT) (residues 1–376) and Alcat1 truncated mutants were expressed in MLE cells for 48 h. The cells were treated with leupeptin for 6 h. Cell lysates were analyzed by immunoblotting for V5 or β-actin. (C) WT V5–Alcat1, V5–K183A or V5-K232A Alcat1 mutants were expressed in cells for 48 h, and cells were then treated with CHX for various times. Cell lysates were subjected to immunoblotting for V5 and β-actin. (D) WT Alcat1 or the K183A mutant were co-expressed with HA–ubiquitin (Ub-HA) in cells for 48 h. Equal amounts of cell lysate (1 mg total protein) were subjected to immunoprecipitation with an antibody against V5; the precipitates were analyzed by immunoblotting for ubiquitin (Ub). The input is 5% of the total cell lysate. (E) WT V5–Alcat1 or a V5–K183A mutant was expressed in cells for 48 h. Equal amounts of cell lysate (1 mg total protein) were subjected to immunoprecipitated with an antibody against ubiquitin; the precipitates were analyzed by immunoblotting for V5. (F,G) Pull-down assays were conducted with truncated mutants to map the region within Alcat1 with which Fbxo28 interacts. Precipitates were then immunoblotted for V5 or Fbxo28 (O28).