Figure 6.
Polycations can substitute for SCF. (A) Polylysine activates Cdc34ΔC autoubiquitination. Reactions containing Cdc34ΔC, ATP, ubiquitin, and E1 enzyme were adjusted to either 10 mm lysine (lane 1), 0.01, 0.1, or 1% polyethylene glycol (PEG, lanes 2–4), or 0.01% (∼300 nm) polylysine (lane 5), and incubated at 20°C for 60 min. Reactions were evaluated as described for Fig. 5A. (B) Polyethyleneimine is an E3 for Gcn4. 35S labeled Gcn4 produced by in vitro translation in reticulocyte lysate and enriched by DEAE chromatography was mixed with E1, Cdc34, ATP, the indicated ubiquitin derivatives (lanes 4–6; H6 stands for His6) and 0.0005% polyethyleneimine (PEI), and incubated at 25°C for 60 min. Reactions conducted in the absence of either Cdc34, PEI, or ubiquitin are depicted in lanes 1–3. Gcn4 was visualized by SDS-PAGE followed by autoradiography. (C) Fusion of Sic1 to Cdc34 does not activate its ubiquitination. Cdc34ΔC–Sic1 (arrowhead) was mixed with E1, ubiquitin, and ATP in the presence of immobilized SCFCdc4 (lane 1), Cdc53PyHA (lane 2), or (as a negative control) Skp1HA (lane 3). Samples were incubated and processed as described for Fig. 5A.