FIGURE 5:
L7 mutations cause a delay in translocon gating that is suppressed by the I86T mutation. (A) The Suc2 series of UTA reporters consist of 1) the N-terminal signal sequence of Suc2p (black, Suc2p1–19); 2) 14- to 296-residue spacer segments (cyan); 3) a Ub domain (red); 4) a 42-residue linker (blue) with a processing site (arrowhead) for a UbP; and 5) a Ura3 reporter domain followed by a triple-HA tag (yellow). (B) After RNCs dock onto the Sec61 complex, the UTA reporter is cleaved if the Ub domain folds in the cytosol but remains intact if translocon gating occurs before the Ub domain emerges from the polypeptide exit tunnel on the large ribosomal subunit. (C, E) In vivo cleavage of the Suc2 (C) and Dap2 (E) reporters. Labels designate the intact reporter (e.g., 23) and cleaved (U-HA) reporter domain. (D, F) Spacer-length dependence of Suc2 (D) and Dap2 (F) reporter cleavage (percentage cytosolic Ura3-HA) in wild-type and mutant strains was calculated after quantification of intact and cleaved forms of the reporters. Symbols are the averages of two or three experiments for each strain as noted, with color-coded error bars designating either the SD or the individual data points.