Fig 4. Pop1 and Pop2 were responsible for the stability of Rev1.
A, pop1 and pop2 deletion mutants stabilized Rev1 protein. wt, pop1Δ, and pop2Δ strains harboring flag-tagged rev1 and an untagged rev1 wt strain were grown, and whole cell extracts were prepared by the boiling method. Protein expression levels were compared by western blotting. The upper panel shows the amount of Rev1 in rev1flag, rev1flag pop2Δ, rev1flag pop1Δ and untagged rev1 strains. The lower panel shows Cdc2 as a loading control. B, Rev1 was coprecipitated with Pop1. rev1flag pop1V5 and rev1flag strains were grown, and whole cell extracts were prepared by the LiNi method. Immunoprecipitation was performed using anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panels show Rev1 and Pop1 in anti-V5-immunoprecipitated fractions. C, Rev1 was co-precipitated with Pop2. The rev1flag pop2V5 strain was grown, and whole cell extracts were prepared. Immunoprecipitation was performed using rabbit normal IgG or anti-V5 antibodies. The left panels show Rev1 and Pop2 input. The right panels show Rev1 and Pop2 in rabbit normal IgG- or anti-V5-immunoprecipitated fractions. D, The Rev1dK mutant was not coprecipitated with Pop1. rev1flag pop1V5 and rev1dKflag pop1V5 strains were grown, and whole cell extracts were prepared. Immunoprecipitation was performed using anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panel shows Rev1 and Pop1 in anti-V5-immunoprecipitated fractions. E, Rev1KK (Rev1 761–818) associated with Pop1. Amino acids 761–818 of Rev1, which is the region deleted in Rev1dKK, were cloned into a pCAM1-flag expression vector. The pop1V5 strain with the pCAM1-rev1KKflag expression vector was grown at 30°C and whole cell extracts were prepared. Immunoprecipitation was performed using rabbit normal IgG or anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panels show Rev1KK and Pop1 in rabbit normal IgG- or anti-V5-immunoprecipitated fractions.