The Skp1 protein from Toxoplasma is modified by a cytoplasmic prolyl 4-hydroxylase associated with oxygen sensing in the social amoeba Dictyostelium.
In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O(2) and α-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans these enzymes control the stability of the HIFα family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O(2) regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3(SCF) ubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1 hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O(2) sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O(2) levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O(2) requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate α-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O(2). The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O(2) by detecting the levels of O(2)-regulated metabolites such as α-ketoglutarate.
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Yuechi
Y
Xu
Kevin M
KM
Brown
Zhuo A
ZA
Wang
Hanke
H
van der Wel
Crystal
C
Teygong
Dongmei
D
Zhang
Ira J
IJ
Blader
Christopher M
CM
West
D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D018503:Genome, Protozoan / Q000502:physiology; D006031:Glycosylation; D006900:Hydroxylation / Q000502:physiology; D007656:Ketoglutaric Acids / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D014122:Toxoplasma / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D007656:Ketoglutaric Acids; D015800:Protozoan Proteins; D044786:S-Phase Kinase-Associated Proteins; D014157:Transcription Factors; D011393:Procollagen-Proline Dioxygenase; D010100:Oxygen