Abstract
Skp1 is a cytoplasmic and nuclear protein of eukaryotes best known as an adaptor in SCF ubiquitin-protein isopeptide ligases. In Dictyostelium, Skp1 is subject to 4-hydroxylation at Pro(143) and subsequent O-glycosylation by alpha-linked GlcNAc and other sugars. Soluble cytosolic extracts have Skp1 prolyl 4-hydroxylase (P4H) activity, which can be measured based on hydroxylation-dependent transfer of [(3)H]GlcNAc to recombinant Skp1 by recombinant (Skp1-protein)-hydroxyproline alpha-N-acetyl-d-glucosaminyltransferase. The Dictyostelium Skp1 P4H gene (phyA) was predicted using a bioinformatics approach, and the expected enzyme activity was confirmed by expression of phyA cDNA in Escherichia coli. The purified recombinant enzyme (P4H1) was dependent on physiological concentrations of O(2), alpha-ketoglutarate, and ascorbate and was inhibited by CoCl(2), 3,4-dihydroxybenzoate, and 3,4-dihydroxyphenyl acetate, as observed for known animal cytoplasmic P4Hs of the hypoxia-inducible factor-alpha (HIFalpha) class. Overexpression of phyA cDNA in Dictyostelium yielded increased enzyme activity in a soluble cytosolic extract. Disruption of the phyA locus by homologous recombination resulted in loss of detectable activity in extracts and blocked hydroxylation-dependent glycosylation of Skp1 based on molecular weight analysis by SDS-PAGE, demonstrating a requirement for P4H1 in vivo. The sequence and functional similarities of P4H1 to animal HIFalpha-type P4Hs suggest that hydroxylation of Skp1 may, like that of animal HIFalpha, be regulated by availability of O(2), alpha-ketoglutarate, and ascorbate, which might exert novel control over Skp1 glycosylation.
Highlights
Skp1 is a subunit of the SCF (Skp1-cullin-F box protein complex) class of ring finger ubiquitin-protein isopeptide ligases (E3),1 which are involved in the polyubiquitination of selected, usually phosphorylated proteins for degradation [1]
Detection and Partial Purification of Skp1 prolyl 4-hydroxylase (P4H) Activity—To search for Skp1 P4H activity in the cytosol of Dictyostelium, assay conditions were adapted from studies on collagen-type and hypoxia-inducible factor (HIF)␣-type P4Hs [26, 27, 38]
GnT1, the polypeptide ␣-GlcNAc-transferase that normally modifies Skp1 hydroxylated at Pro143, was expressed in E. coli as rGnT1, partially purified, and added with UDP-[3H]GlcNAc after 2 h of incubation, and the reaction was continued for 2 h. rGnT1 was not inhibited by the P4H cosubstrates and cofactors in control experiments using Skp1A1-Myc purified from Dictyostelium as the acceptor substrate
Summary
E3, ubiquitin-protein isopeptide ligase; GnT1, (Skp protein)-hydroxyproline ␣-N-acetyl-D-glucosaminyltransferase; HIF, hypoxia-inducible factor; P4H, prolyl 4-hydroxylase; VHL, von Hippel-Lindau; r, recombinant; mAb, monoclonal antibody; TEV, tobacco etch virus. The Dictyostelium sequence most closely related to the diatom and oomycete P4H-like sequence, implicated in the Skp modification pathway by its association with an Skp GnT1-like sequence, was the most closely related of the five to the HIF␣ class of animal P4H sequences This gene, referred to as phyA, is shown here to encode Skp P4H based on its biochemical activity and its requirement for Skp glycosylation in vivo. This finding in the model system Dictyostelium suggests that HIF␣ P4Hs are an ancient enzyme family that had its evolutionary origins in lower eukaryotes or even prokaryotes and opens new lines of studies into O2 and metabolite regulation of cell physiology in microbes, including parasites and pathogens of plants and animals
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