FIGURE 5.

Relationship between AgtA and other Skp1 interactors. A, effect of AgtA on Skp1 PhyA and Gnt1 reactions. Reactions containing 0.1 μm unmodified Skp1 (left panel) or HO-Skp1 (right panel) were supplemented with 0, 0.1, or 0.5 μm His₆AgtA-FL or His₆AgtA(D132A) as indicated. B, effect of Fbs1 on AgtA-mediated α-galactosylation of Skp1. FGGn-Skp1 (0.5 μm) was preincubated with 0–50 μm His₆Fbs1, and its α-galactosylation was tested as described in the legend to Fig. 2A. C, effect of Fbs1 on Skp1 activation of AgtA activity. AgtA modification of 1 mm F-pNP was measured in the presence of 100 nm unmodified (light gray) or α-GlcNAcylated (dark gray) Skp1 (as in Fig. 3A), 10 μm UDP-[³H]Gal, and the indicated concentration of Fbs1, for 30 min. Data are presented as mean ± S.D. for assays conducted in triplicate, and are representative of at least 2 independent trials.