Figure 4

The synthesis of free polyubiquitin chains in vitro and in vivo. (A). Regulation of free polyubiquitin chain synthesis by substrate recruitment to SCF^Cdc4. 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 Ub_1-6 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 Ubp15^C354A supports growth of ^tm CDC34 upb14 Δ yeast. Heterozygous diploids (RC171 and RC172) were transformed with the indicated plasmids that overexpress Ubp14 (pUBP14) or Ubp14^C354A (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.