Analysis of Skp1 glycosylation and nuclear enrichment in Dictyostelium

Skp1 is a subunit of SCF-E3 ubiquitin ligases and other protein complexes i n the nucleus and cytoplasm of yeast and mammalian cells. In Dictyostelium, Skp1 is partially modified by an unusual pentasaccharide O-linked to hydro xyproline143. This modification was found to be susceptible to known prolyl hydroxylase inhibitors based on M-r-shift analysis using SDS-polyacrylamid e gel electrophoresis/Western blotting, In addition, Dictyostelium Skp1 con sists of 2 genetic isoforms, Skp1A and Skp1B, which differ by a single amin o acid and appear to be expressed throughout the life cycle based on revers e-transcription polymerase chain reactions, The significance of these struc tural variations was examined by expressing myc-tagged Skp1s and mutants th at lacked the glycosylation site. Gel-based M-r-shift studies show ed that Skp1A and Skp1B are both nearly completely glycosylated during growth and e arly development, and mass spectrometry of glycopeptides showed that they w ere glycosylated similarly, Skp1 expressed later in prespore cells was not glycosylated, unlike bulk Skp1 persisting from earlier in development, but became glycosylated after return to growth medium. Skp1A and Skp1B were eac h concentrated in the nucleus and regions of the cytoplasm, based on immuno fluorescence localization, However, when Skp1 glycosylation was blocked by mutation, prolyl hydroxylase inhibitors, or expression in prespore cells, n uclear concentration of Skp1 was not detected. Furthermore, nuclear concent ration occurred in a mutant that attached only the core disaccharide to Skp 1, Overall, there was no evidence for differential Skp1 isoform expression, glycosylation variants in the bulk Skp1 pool, or regulation of nuclear loc alization. However, these studies uncovered evidence that the glycosylation pathway is developmentally regulated and can function posttranslationally, and that core glycosylation is required for Skp1's nuclear concentration.