Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium.

O(2) regulates multicellular development of the social amoeba Dictyostelium, suggesting it may serve as an important cue in its native soil environment. Dictyostelium expresses an HIFα-type prolyl 4-hydroxylase (P4H1) whose levels affect the O(2)-threshold for culmination implicating it as a direct O(2)-sensor, as in animals. But Dictyostelium lacks HIFα, a mediator of animal prolyl 4-hydroxylase signaling, and P4H1 can hydroxylate Pro143 of Skp1, a subunit of E3(SCF)ubiquitin-ligases. Skp1 hydroxyproline then becomes the target of five sequential glycosyltransferase reactions that modulate the O(2)-signal. Here we show that genetically induced changes in Skp1 levels also affect the O(2)-threshold, in opposite direction to that of the modification enzymes suggesting that the latter reduce Skp1 activity. Consistent with this, overexpressed Skp1 is poorly hydroxylated and Skp1 is the only P4H1 substrate detectable in extracts. Effects of Pro143 mutations, and of combinations of Skp1 and enzyme level perturbations, are consistent with pathway modulation of Skp1 activity. However, some effects were not mirrored by changes in modification of the bulk Skp1 pool, implicating a Skp1 subpopulation and possibly additional unknown factors. Altered Skp1 levels also affected other developmental transitions in a modification-dependent fashion. Whereas hydroxylation of animal HIFα results in its polyubiquitination and proteasomal degradation, Dictyostelium Skp1 levels were little affected by its modification status. These data indicate that Skp1 and possibly E3(SCF)ubiquitin-ligase activity modulate O(2)-dependent culmination and other developmental processes, and at least partially mediate the action of the hydroxylation/glycosylation pathway in O(2)-sensing.