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.

FIGURE 1.

Modifications of gnt1 locus. A, genomic locus of gnt1 from D. discoideum. Positions of oligonucleotides used for cloning and sequencing (see supplemental Table S1 and supplemental Fig. S1) are shown. B, corresponding coding region of gnt1 from D. purpureum. C, hybrid DdDpGnt1.b (gnt1.0) resulting from gene replacement strategy shown in panel D. D, gene replacement constructs designed for homologous recombination based on a double-crossover mechanism, after selection based on resistance to hygromycin. Left, 5′-targeting DNA including most of exon-2 of Dd-gnt1; right, 3′-targeting DNA including the coding region of the downstream gene. Replacement DNA-1 contains the inactivating H104D substitution (termed gnt1.1); replacement DNA-2 contains, in addition, Y253stop (gnt1.3). E and F, complementation constructs consisting of the coding regions of DpGnt1 or mutant DpGnt1(D102A) (gnt1.2), positioned downstream of the ecmA promoter and adjacent to a G418 resistance cassette; transformants are expected to overexpress Gnt1 in prestalk cells from chromosomally integrated multicopy tandem arrays. G, PCR analysis of gene replacement strains. PCR reactions conducted on genomic DNA from strains transformed with DNA-1 or -2, using the indicated primer pairs, one from outside and the other from within the disruption DNA. Std, DNA ladder standard.

FIGURE 2.

Skp1-Hyp GlcNAcT activity in gnt1 mutant strains. Soluble (S100) extracts were prepared from slugs of normal and mutant strains and desalted, and ∼35 μg of protein was incubated with 2 μm recombinant HO-Skp1 in the presence of UDP-[³H]GlcNAc. Incorporation of ³H was measured using the SDS-PAGE assay, and the zero time blank value was subtracted. Error bars indicate ± S.E.

FIGURE 3.

Skp1 modification status in gnt1 mutant strains. A, summary of the Skp1 modification pathway. B, isoform-specific antibodies. Soluble (S100) extracts of growing cells of normal (Ax3 = wild type) and mutant strains were subjected to SDS-PAGE/Western blotting with pan-specific mAb 4E1, the previously described GlcNAc-O-Skp1-specific mAb 1C9, and two new isoform-selective pAbs UOK87 and UOK85. The series of mutant strains shown express incrementally modified Skp1 isoforms from left to right as indicated (Pro, unmodified; OH, hydroxylated; Gn, modified with GlcNAc; G, Gal-GlcNAc; F, Fuc-Gal-GlcNAc; or GG′, Gal-Gal-Fuc-Gal-GlcNAc). C, slug stage extracts of gnt1 mutant and overexpression strains were Western blotted using the indicated antibodies to assess the modification status of Skp1 and expression of DpGnt1. Note: The appearance of Skp1 as a doublet in these trials resulted from apparent proteolysis that occurs variably during preparation of slug cell extracts. D, MALDI-TOF-MS analysis of Skp1 isolated from gnt1.1 stationary stage cells. Skp1 was purified to homogeneity from a 30-liter culture of gnt1.1 cells and analyzed by MALDI-TOF-MS.

FIGURE 4.

O₂ dependence of culmination of gnt1 mutant cells. A, gnt1 mutant strains, in parallel with normal (Ax3) and complemented strains, were allowed to culminate following consumption of bacteria on SM-agar and photographed from above. Typical fruiting bodies, consisting of a spherical droplet of spores perched on an upright, linear stalk, are circled in yellow. B, axenically grown gnt1 mutant strains, in parallel with Ax3 and phyA⁻ cells, were induced to develop for 42 h on filters at 10 or 15% O₂. C, spores were recovered from trials like those shown in panel B and counted in a hemacytometer. Spore numbers were normalized to the maximal number formed at 40 or 21% O₂, which varied ±20%. Sporulation corresponded to culmination in these strains. Data are merged from two large trials and are representative of observations in seven independent trials. D, strains in which Gnt1 alleles were overexpressed under control of the ecmA promoter were analyzed as in panel C. E, average O₂ values (± S.E.) required for 50% sporulation from the seven trials are plotted.

FIGURE 5.

Gnt1 substrate detection by in vitro complementation. A, soluble extracts from growing gnt1.1 and normal (Ax3) cells were incubated with UDP-[³H]GlcNAc ± recombinant DpGnt1. The quenched reaction was subjected to SDS-PAGE, and the apparent M_r distribution of ³H incorporation was determined by liquid scintillation counting of sequential gel slices. B, replot of the same data using a different ordinate scale.

FIGURE 6.

Effect of prolyl hydroxylation on Skp1 stability. phyA⁻ (P4H1⁻) and gnt1.1 cells were compared in 21% O₂. A, proliferating cells were labeled with low specific activity [³⁵S]Met/Cys for 1 h and incorporation into total protein determined by trichloroacetic acid precipitation. Average values ± S.E. are shown. B, proliferating cells were labeled with high specific activity [³⁵S]Met/Cys for 1 h, and incorporation into Skp1 was determined by immunoprecipitation and SDS-PAGE. dpm were determined by scintillation counting and are presented as the percentage of incorporation into total protein. C, a comparison of labeling of slug stage Skp1, normalized to input cell number. D, half-lives of proliferating cell Skp1 were calculated from data shown in panel E. E, results from a pulse-chase study of 1-h labeled proliferating cells are plotted as a function of time. The line was fitted from a linear regression analysis and used to calculated Skp1 half-lives in growing cells. The autoradiogram at the left shows the selectivity of the immunoprecipitation method. Less incorporation was observed into the phyA⁻ strain in the example shown, but the fraction of label relative to total incorporation was similar between the two strains.