FIG. 7.
Smt3-protein conjugates in ulp2Δ cells. (A) Effect on Smt3-protein conjugates of overproduction of Ulp2 or Ulp1. vector, pRS424; all alleles are in pRS424. The arrowheads indicate prominent Smt3 conjugates that accumulate in ulp2Δ cells. Samples were run on an SDS–7.5 to 18% polyacrylamide gradient gel followed by anti-Smt3 immunoblotting. Free Smt3 was not detected under the blotting conditions used. (B) Cleavage of Smt3-protein conjugates in yeast extracts. Spheroplasts were incubated at 37°C for 30 min and then lysed with (+) or without (−) NEM. After 30 min at 30°C, 90 μg of protein from each sample was loaded on a 10 to 15% gradient gel, followed by immunoblot analysis. The asterisk and bracket denote Smt3 conjugates from ulp1ts cells that, unlike most of the conjugates, disappeared during in vitro incubation without NEM. (C) Smt3-precursor processing analyzed by anti-Smt3 immunoblotting with enhanced chemiluminescent detection. The strains used were MHY1614 to MHY1617 that expressed SMT3 from a plasmid-borne CUP1 promoter.