title: Skp1 prolyl 4-hydroxylase of dictyostelium mediates glycosylation-independent and -dependent responses to O2 without affecting Skp1 stability.
abstract: Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O(2) sensing in animals via modification of the transcriptional factor subunit HIFα, resulting in its polyubiquitination by the E3(VHL)ubiquitin (Ub) ligase and degradation in the 26 S proteasome. Previously thought to be restricted to animals, a homolog (P4H1) of HIFα-type PHDs is expressed in the social amoeba Dictyostelium where it also exhibits characteristics of an O(2) sensor for development. Dictyostelium lacks HIFα, and P4H1 modifies a different protein, Skp1, an adaptor of the SCF class of E3-Ub ligases related to the E3(VHL)Ub ligase that targets animal HIFα. Normally, the HO-Skp1 product of the P4H1 reaction is capped by a GlcNAc sugar that can be subsequently extended to a pentasaccharide by novel glycosyltransferases. To analyze the role of glycosylation, the Skp1 GlcNAc-transferase locus gnt1 was modified with a missense mutation to block catalysis or a stop codon to truncate the protein. Despite the accumulation of the hydroxylated form of Skp1, Skp1 was not destabilized based on metabolic labeling. However, hydroxylation alone allowed for partial correction of the high O(2) requirement of P4H1-null cells, therefore revealing both glycosylation-independent and glycosylation-dependent roles for hydroxylation. Genetic complementation of the latter function required an enzymatically active form of Gnt1. Because the effect of the gnt1 deficiency depended on P4H1, and Skp1 was the only protein labeled when the GlcNAc-transferase was restored to mutant extracts, Skp1 apparently mediates the cellular functions of both P4H1 and Gnt1. Although Skp1 stability itself is not affected by hydroxylation, its modification may affect the stability of targets of Skp1-dependent Ub ligases.
authors: Department of Biochemistry and Molecular Biology, 975 NE 10th St., BRC 417, OUHSC, Oklahoma City, OK 73104, USA.
authors: Dongmei
authors: D
authors: Zhang
authors: Hanke
authors: H
authors: van der Wel
authors: Jennifer M
authors: JM
authors: Johnson
authors: Christopher M
authors: CM
authors: West
mesh_terms: D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability / Q000502:physiology; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D006900:Hydroxylation / Q000502:physiology; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D006899:Mixed Function Oxygenases; D016695:Glycosyltransferases; D010100:Oxygen