Skp1 Prolyl 4-Hydroxylase of Dictyostelium Mediates Glycosylation-independent and -dependent Responses to O2 without Affecting Skp1 Stability

Dongmei Zhang,Hanke Van Der Wel,Jennifer M Johnson,Christopher M West

doi:10.1074/jbc.m111.314021

Dongmei Zhang, Hanke Van Der Wel + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m111.314021

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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.

Highlights

GlcNAc capping confounds analysis of prolyl 4-hydroxylation by P4H1 in Dictyostelium O2 sensing
Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O2 sensing in animals via modification of the transcriptional factor subunit HIF␣, resulting in its polyubiquitination by the E3VHLubiquitin (Ub) ligase and degradation in the 26 S proteasome
The C-terminal 143 amino acids of Gnt1, which include a non-conserved degenerate 79-amino acid domain highly enriched in Asn residues that interfere with cloning and expression in Escherichia coli [14], were replaced with the corresponding 71-amino acid region of Gnt1 from the related species D. purpureum [25] that lacks the degenerate region (Fig. 1B)

Summary

Background

GlcNAc capping confounds analysis of prolyl 4-hydroxylation by P4H1 in Dictyostelium O2 sensing. Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O2 sensing in animals via modification of the transcriptional factor subunit HIF␣, resulting in its polyubiquitination by the E3VHLubiquitin (Ub) ligase and degradation in the 26 S proteasome. Hydroxylation alone allowed for partial correction of the high O2 requirement of P4H1-null cells, revealing both glycosylation-independent and glycosylation-dependent roles for hydroxylation Genetic complementation of the latter function required an enzymatically active form of Gnt. Hydroxylation partially rescued the high O2 requirement of the P4H1 mutant, and Skp was the only substrate of Gnt that could be detected in gnt1-null cells, suggesting an alternative primordial mechanism of hydroxylation that might still affect the stability of clients of Skp1-dependent Ub ligases

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DISCUSSION