Prolyl 4-hydroxylase-1 mediates O2 signaling during development of<i>Dictyostelium</i>

West, Christopher M.; Wel, Hanke van der; Wang, Zhuo A.

doi:10.1242/dev.000893

Cited by 61 publications

(143 citation statements)

References 50 publications

(72 reference statements)

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“…Dictyostelium Growth and Development-Cells were grown axenically in HL-5 medium, and assayed for O 2 -dependent development as described previously (19). Briefly, cells were deposited on filters in PDF buffer and developed in the dark for 42 h under the indicated O 2 concentrations with the balance made up with N 2 .…”

Section: Methodsmentioning

confidence: 99%

Novel Regulation of Skp1 by the Dictyostelium AgtA α-Galactosyltransferase Involves the Skp1-binding Activity of Its WD40 Repeat Domain

Schafer

Sheikh

Zhang³

et al. 2014

Journal of Biological Chemistry

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Background: AgtA mediates terminal ␣-galactosylation of Skp1, but genetic studies show an unexpectedly profound role in oxygen sensing. Results: AgtA exhibits salt-sensitive Skp1-binding activity that competes with other Skp1 interactions but subsequently activates ␣-galactosylation that alters Skp1 binding. AgtA regulates development through non-enzymatic mechanisms. Conclusion: AgtA modulates Skp1 interactions by a self-limiting glycosylation mechanism. Significance: Polypeptide recognition by a glycosyltransferase mediates non-enzymatic functions.

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Section: Methodsmentioning

confidence: 99%

Novel Regulation of Skp1 by the Dictyostelium AgtA α-Galactosyltransferase Involves the Skp1-binding Activity of Its WD40 Repeat Domain

Schafer

Sheikh

Zhang³

et al. 2014

Journal of Biological Chemistry

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“…The culE 5Ј-targeting sequence was excised via digestion with BssHII and BglII (italicized) and ligated into similarly digested pVS3 vector to yield pVSCulE5Ј. The pVS3 vector series (pVS3, pVS3C, pVS3E) was derived from pVS (18), pVSC (9), and pVSE (9), which differ in their discoidin 1␥, cotB, and ecmA promoters, respectively, through replacement of their myc tags with a sequence that encoded the following motifs: ATG/BclI/His 6 /BglII/3ϫFLAG/EcoRI/BirA/NcoI/TEV protease site/BamHI/TAA. We achieved this by cutting the pVS vectors with KpnI and BamHI and through replacement by ligating the following synthetic sequence annealed from complementary oligonucleotides and digested with the same restriction enzymes: GGTACCAAAAAA-ATGTCTGATCATCATCATCATCATCATAGATCTGATTATAAGGATG-ATGATGATAAGGATTATAAGGATGATGATGATAAGGATTATAAGGA-TGATGATGATAAGGAATTCGGTGGTGGTTTAAATGATATTTTTGAA-GCACAAAAAATTGAATGGCATATCCATGGTGAAAACTTGTATTTCC-AAGGTGGATCC.…”

Section: Metabolic Labeling and Isolation Of Skp1-cellsmentioning

confidence: 99%

“…The FbxD DNA was excised via digestion with BamHI and SacI (italicized in primer sequences) and ligated into a similarly digested pVSC vector (9), which inserted an amino-terminal FLAG tag and directs expression in prespore cells under control of the cotB promoter. Cells were transformed as described above in the presence of 10 g/ml G-418 (Research Products International, Mount Prospect, IL) in HL-5ϩ medium, further selected with 20 or 120 g/ml G-418 in HL-5 in gyrating flasks, and cloned as described above.…”

Section: Metabolic Labeling and Isolation Of Skp1-cellsmentioning

confidence: 99%

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Glycosylation of Skp1 Promotes Formation of Skp1–Cullin-1–F-box Protein Complexes in Dictyostelium

Sheikh¹,

Xu²,

Wel³

et al. 2015

Molecular & Cellular Proteomics

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O 2 sensing in diverse protozoa depends on the prolyl 4 hydroxylation of Skp1 and modification of the resulting hydroxyproline with a series of five sugars. In yeast, plants, and animals, Skp1 is associated with F-box proteins. The Skp1-F-box protein heterodimer can, for many F-box proteins, dock onto cullin-1 en route to assembly of the Skp1-cullin-1-F-box protein-Rbx1 subcomplex of E3 SCF Ub ligases. E3 SCF Ub ligases conjugate Lys48-polyubiquitin chains onto targets bound to the substrate receptor domains of F-box proteins, preparing them for recognition by the 26S proteasome. In the social amoeba Dictyostelium, we found that O 2 availability was rate-limiting for the hydroxylation of newly synthesized Skp1. To investigate the effect of reduced hydroxylation, we analyzed knockout mutants of the Skp1 prolyl hydroxylase and each of the Skp1 glycosyltransferases. Proteomic analysis of co-immunoprecipitates showed that wild-type cells able to fully glycosylate Skp1 had a greater abundance of an SCF complex containing the cullin-1 homolog CulE and FbxD, a newly described WD40-type F-box protein, than the complexes that predominate in cells defective in Skp1 hydroxylation or glycosylation. Similarly, the previously described FbxA-Skp1CulA complex was also more abundant in glycosylation-competent cells. The CulE interactome also included higher levels of proteasomal regulatory particles when Skp1 was glycosylated, suggesting increased activity consistent with greater association with F-box proteins. Finally, the interactome of FLAG-FbxD was modified when it harbored an F-box mutation that compromised Skp1 binding, consistent with an effect on the abundance of potential substrate proteins. We Timely protein degradation is a cornerstone of cell cycling and the regulation of numerous physiological and developmental processes. Eukaryotes have evolved an extensive array of polyubiquitination enzymes to tag proteins on a proteinby-protein basis as a recognition marker for degradation in the 26S proteasome. The cullin-RING ubiquitin ligases (CRLs) 1 are a prominent subgroup of these enzymes (1) and consist of an E3 architecture that includes a substrate receptor, an adaptor (in most cases), the cullin scaffold, the RING protein, and an exchangeable E2 ubiquitin donor that has been charged with ubiquitin (Ub) by an E1 enzyme. The first discovered and still prototypic example is the CRL1 class (2), also referred to as SCF on account of the names of its founding subunits, Skp1, cullin-1, and F-box proteins (FBPs). The CRL1 (or SCF) complexes utilize FBPs as substrate receptors, Skp1 as the adaptor linking the FBP to the N-terminal region of cullin-1 (Cul1), and Rbx1 as the RING protein that tethers the E2 Ub donor to the Cul1 C-terminal region (see Fig. 2B). CRL1s can be activated by neddylation of Cul1 by a Nedd8-specific E2, which mobilizes Rbx1 to afford rotational flexibility of the E2 and displaces the inhibitor Cand1, permitting docking of the Skp1-FBP heterodimer (3-5). Deneddylation mediated by the eight-subunit COP...

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“…Samples were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to Western blot analysis as described previously (42). Lanes contained 5 ϫ 10 5 spores, slug cells, or the equivalent amount of ISM.…”

Section: Methodsmentioning

confidence: 99%

Dependence of Stress Resistance on a Spore Coat Heteropolysaccharide in Dictyostelium

West¹,

Nguyen²,

Wel³

et al. 2009

Eukaryot Cell

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In Dictyostelium, sporulation occurs synchronously as prespore cells approach the apex of the aerial stalk during culmination. Each prespore cell becomes surrounded by its own coat comprised of a core of crystalline cellulose and a branched heteropolysaccharide sandwiched between heterogeneous cysteine-rich glycoproteins. The function of the heteropolysaccharide, which consists of galactose and N-acetylgalactosamine, is unknown. Two glycosyltransferase-like genes encoding multifunctional proteins, each with predicted features of a heteropolysaccharide synthase, were identified in the Dictyostelium discoideum genome. pgtB and pgtC transcripts were modestly upregulated during early development, and pgtB was further intensely upregulated at the time of heteropolysaccharide accumulation. Disruption of either gene reduced synthase-like activity and blocked heteropolysaccharide formation, based on loss of cytological labeling with a lectin and absence of component sugars after acid hydrolysis. Cell mixing experiments showed that heteropolysaccharide expression is spore cell autonomous, suggesting a physical association with other coat molecules during assembly. Mutant coats expressed reduced levels of crystalline cellulose based on chemical analysis after acid degradation, and cellulose was heterogeneously affected based on flow cytometry and electron microscopy. Mutant coats also contained elevated levels of selected coat proteins but not others and were sensitive to shear. Mutant spores were unusually susceptible to hypertonic collapse and damage by detergent or hypertonic stress. Thus, the heteropolysaccharide is essential for spore integrity, which can be explained by a role in the formation of crystalline cellulose and regulation of the protein content of the coat.In Dictyostelium, spores are the only surviving cell type produced by starvation-induced multicellular development. During this process, solitary amoebae aggregate to form a migratory slug composed of prespore and prestalk cells, which then culminate to form a fruiting body consisting of spores perched on top of a 1 to 2 mm tall cellular stalk. As each prespore cell becomes a spore, it dehydrates, accumulates trehalose, and encloses itself in a specialized cell wall (37). This physicochemical barrier protects the enclosed amoeba from external stress and probably actively regulates terminal sporulation and spore germination. The spore coat is formed de novo from four known sources: (i) an early-formed pool of proteins and (ii) a galactose-rich polysaccharide (GPS) stored together in prespore vesicles (PSVs) of the slug, (iii) a late-formed pool of protein(s), including SP65 (23), and (iv) cellulose formed de novo at the cell surface. These components are separately deposited at the cell surface, where they organize into an asymmetrical trilaminar "sandwich" with proteins on either side enclosing the polysaccharides in the interior (see Fig. 5B).Cellulose is the primary structural component of the middle layer and is required for organization of the prote...

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Prolyl 4-hydroxylase-1 mediates O2 signaling during development ofDictyostelium

Cited by 61 publications

References 50 publications

Novel Regulation of Skp1 by the Dictyostelium AgtA α-Galactosyltransferase Involves the Skp1-binding Activity of Its WD40 Repeat Domain

Novel Regulation of Skp1 by the Dictyostelium AgtA α-Galactosyltransferase Involves the Skp1-binding Activity of Its WD40 Repeat Domain

Glycosylation of Skp1 Promotes Formation of Skp1–Cullin-1–F-box Protein Complexes in Dictyostelium

Dependence of Stress Resistance on a Spore Coat Heteropolysaccharide in Dictyostelium

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