Conjugation of Rub1 to Cdc53 in vitro depends on Rbx1. Total cell lyaste (200 μg) from Sf21 cells infected with the indicated baculoviruses was immunoprecipitated with 2 μg of anti-Cdc53(yN-18) antibody. Immunoprecipitates were assayed for Rub1 conjugation activity at 25°C for 30 min, and reaction products were analyzed by immunoblotting with anti-Cdc53(yC-17), anti-GST, or anti-myc antibodies.

Conjugation of Rub1 to Cul2 depends on Rbx1. (A) Cell lysate (20 μg) from the indicated baculovirus-infected Sf21 cells was immunoblotted with anti-HA or anti-myc antibodies. Total cell lyaste protein (200 μg) from baculovirus-infected Sf21 cells was immunoprecipitated with 2 μg of anti-myc antibody. The beads were incubated with 30 ng of Ula1, 20 ng of Uba3, 100 ng of Ubc12, 1 μg of GST–Rub1, and 1.5 mm ATP at 25°C for 50 min and immunoblotted with ant-HA or anti-GST antibodies. (B) After immunoprecipitation with 12 μg of anti-myc antibody from 1.2 mg of baculovirus infected Sf21 cell lysate, the beads were divided into six tubes and incubated at 25°C for 30 min in the presence or absence of Uba3/Ubc12, GST–Rub1, and ATP as indicated and immunoblotted with anti-HA or anti-GST antibodies.

Conjugation of Rub1 to Cdc53 in vitro. (A) Total cell lysate (20 μg) from Sf21 cells infected with the indicated baculoviruses was immunoblotted with anti-Cdc53(yC-17) or anti-myc antibodies. (B) Total cell lysate (200 μg) from baculovirus-infected Sf21 cells was immunoprecipitated with 2 μg of anti-myc antibody and then immunoblotted with anti-Cdc53(yC-17) or anti-myc antibodies. (C) Total cell lysate (800 μg) from baculovirus-infected Sf21 cells was immunoprecipitated with 8 μg of anti-myc antibody. After immunoprecipitation, protein A Sepharose beads were divided into four tubes and and assayed for Rub1 conjugation activity at 25°C for the indicated times. Reaction products were immunoblotted with anti-Cdc53(yC-17) or anti-GST antibodies. (D) Total baculovirus-infected total cell lysate (1200 μg) was immunoprecipitated with 12 μg of anti-myc antibody. After immunoprecipitation, protein A Sepharose beads were divided into six tubes and assayed for Rub1 conjugation activity incubated at 25°C for 30 min in the presence or absence of Uba3/Ula1, Ubc12, GST–Rub1, and ATP as indicated and immunoblotted with anti–Cdc53(yC-17) or anti-GST antibodies. The asterisks (*) indicate a higher molecular mass Cdc53 species that is most likely conjugated to endogenous Rub1.

Mutation of predicted zinc-binding residues of Rbx1 is lethal in S. cerevisiae and interferes with Rbx1 activity in Rub1 conjugation and in ubiqutination of Cln2 by SCF^Grr1. (A) Alignment of Rbx1 from human, Drosophila melanogaster (Dros), C. elegans (Elegans), or S. cerevisiae (Yeast) with APC11 from S. cerevisiae. (B) Effects of Rbx1 mutations on S. cerevisiae viability. (C) Cell lysate (200 μg) from Sf21 cells infected with the indicated baculoviruses was immunoprecipitated with 2 μg of anti-myc antibodies. The immunoprecipitates were assayed at 25°C for 50 min for Rub1 conjugation activity and reaction products were immunoblotted with anti-Cdc53(yC-17) or anti-myc antibodies (top panels). Cell lysate protein (20 μg) from the same cells was immunoblotted with anti-Cdc53(yC-17) or anti-GST antibodies (bottom panels). (D) Total cell lyaste (400 μg) from Sf21 cells infected with the indicated baculoviruses was immunoprecipitated with 4 μg of anti-myc antibody. One-half of the immunoprecipitate was immunoblotted with anti-HSV, anti-Cdc53(yC-17), anti-FLAG, or anti-myc antibodies (middle panels). The remaining immunoprecipitate was assayed for SCF^Grr1 activity at 25°C for 60 min as described in Materials and Methods. Reaction products were immunoblotted with anti-HA antibody (top panel). The same cell lysates (20 μg) were immunoblotted with anti-HSV, anti–Cdc53(yC-17), anti-FLAG, or anti-myc antibodies (bottom panels).