The synthesis of free polyubiquitin chains in vitro and in vivo. (A). Regulation of free polyubiquitin chain synthesis by substrate recruitment to SCFCdc4. Standard ubiquitination reactions with Cdc34 or tmCdc34 (5 pmol) were analyzed by western blots with α-Ub, α-Sic1 or α-Cdc34 antibodies. (B). IsoT sensitivity. Reactions as in A lanes 4 and 8 were analyzed for IsoT sensitivity (Methods). (C). Levels of ubiquitin and ubiquitin conjugates in vivo. Boiled cell extracts (Methods) were analyzed by western blot with α-Ub (Covance) or α-Rpn10 antibodies. Lane 4: 200 ng of free polyubiquitin chains Ub1-6 purchased from Enzo. (D). Growth of tm
CDC34 but not CDC34 yeast is sensitive to loss of UBP14. Haploids with the indicated genotypes were selected on haploid selection media with G418 and nourseothricin at 30°C for three days. (E). Overexpression of Ubp14 but not Ubp15C354A supports growth of tm
CDC34 upb14 Δ yeast. Heterozygous diploids (RC171 and RC172) were transformed with the indicated plasmids that overexpress Ubp14 (pUBP14) or Ubp14C354A (pUBP14-C354A), patched onto sporulation media and incubated at 26°C for five days. Haploids with the indicated genotypes were selected as in D. (F). Accumulation of ubiquitin conjugates in yeast extracts enriched with Uba1, ubiquitin, ATP and MgCl2. Extracts with active ubiquitin-proteasome system (10 μg of total proteins; see Methods) were enriched with Uba1 (10 pmol), ubiquitin (1.3 nmol) ATP (2 mM) and MgCl2 (2 mM), incubated at 25°C for the times indicated and analyzed by 10% or 18% SDS-PAGE followed by α-Ub (Sigma) or α-Rpn10 WB.