title: Glycosylation of Skp1 promotes formation of Skp1-cullin-1-F-box protein complexes in dictyostelium.
abstract: O(2) sensing in diverse protozoa depends on the prolyl 4 hydroxylation of Skp1 and modification of the resulting hydroxyproline with a series of five sugars. In yeast, plants, and animals, Skp1 is associated with F-box proteins. The Skp1-F-box protein heterodimer can, for many F-box proteins, dock onto cullin-1 en route to assembly of the Skp1-cullin-1-F-box protein-Rbx1 subcomplex of E3(SCF)Ub ligases. E3(SCF)Ub ligases conjugate Lys48-polyubiquitin chains onto targets bound to the substrate receptor domains of F-box proteins, preparing them for recognition by the 26S proteasome. In the social amoeba Dictyostelium, we found that O(2) availability was rate-limiting for the hydroxylation of newly synthesized Skp1. To investigate the effect of reduced hydroxylation, we analyzed knockout mutants of the Skp1 prolyl hydroxylase and each of the Skp1 glycosyltransferases. Proteomic analysis of co-immunoprecipitates showed that wild-type cells able to fully glycosylate Skp1 had a greater abundance of an SCF complex containing the cullin-1 homolog CulE and FbxD, a newly described WD40-type F-box protein, than the complexes that predominate in cells defective in Skp1 hydroxylation or glycosylation. Similarly, the previously described FbxA-Skp1CulA complex was also more abundant in glycosylation-competent cells. The CulE interactome also included higher levels of proteasomal regulatory particles when Skp1 was glycosylated, suggesting increased activity consistent with greater association with F-box proteins. Finally, the interactome of FLAG-FbxD was modified when it harbored an F-box mutation that compromised Skp1 binding, consistent with an effect on the abundance of potential substrate proteins. We propose that O(2)-dependent posttranslational glycosylation of Skp1 promotes association with F-box proteins and their engagement in functional E3(SCF)Ub ligases that regulate O(2)-dependent developmental progression.
authors.affiliation: From the ‡Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104;
authors.forename: M Osman
authors.initials: MO
authors.lastname: Sheikh
authors.affiliation: From the ‡Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104;
authors.forename: Yuechi
authors.initials: Y
authors.lastname: Xu
authors.affiliation: From the ‡Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104;
authors.forename: Hanke
authors.initials: H
authors.lastname: van der Wel
authors.affiliation: From the ‡Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104;
authors.forename: Paul
authors.initials: P
authors.lastname: Walden
authors.affiliation: §Department of Biochemistry & Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Hartson
authors.affiliation: From the ‡Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104;
authors.forename: Christopher M
authors.initials: CM
authors.lastname: West
mesh_terms: D004023:Dictyostelium / Q000378:metabolism*; D044783:F-Box Proteins / Q000378:metabolism*; D006031:Glycosylation; D006900:Hydroxylation; D010100:Oxygen / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism*; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism*; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism*
chemical_list: D044783:F-Box Proteins; D015800:Protozoan Proteins; D044786:S-Phase Kinase-Associated Proteins; D044843:SKP Cullin F-Box Protein Ligases; D010100:Oxygen