Fig. 8. Translocation defects of srp14-ΔC29 cells in the UTA assay. (A) Organization of UTA constructs. The protein is depicted N- to C-terminus; signal sequence (SS), spacer (SPACER), ubiquitin (UBI) and Ura3p. The cleavage site for cytosolic ubiquitin-dependent proteases is indicated (arrow). (B and C) srp14-ΔC29 cells streaked on to a –trp (B; selecting for the plasmids) and a –ura (C; selecting for cytosolic Ura3p) plate were incubated for 2 days at 30°C. They contained empty vector (top left), Suc2₂₇₇ (top right), Dap2₃₀₀ (bottom left) or the control substrate Suc2₂₃, which has a 23 amino acid spacer (bottom right). (D and E) As for (B) and (C) but with wild-type cells. As seen previously (Johnsson and Varshavsky, 1994), Suc2₂₃ gave a Ura+ phenotype in wild-type cells. (F) Anti-HA immunoprecipitations from extracts of pulse-labelled cells expressing Suc2₅₁₈UbDHFR_HA (lanes 1–3) or Suc2₂₃UbDHFR_HA control, which only yields the DHFR_HA fragment (lane 4) (Johnsson and Varshavski, 1994). Labelling of cells and analysis are as in Figure 7.