Figure 7.

(A) The β′-COP WD40 domain mutants sec27Δ1-285 and sec27-95 behave like wild-type for KKTN trafficking in vivo. sec27Δ1-285 and sec27-95 cells and ret1Δ1-285 and wild-type (CEN::SEC27) cells as controls, all expressing the Wbp1-Inv.-KKTN fusion construct, were grown at 24°C. Before the experiment cells were preshifted to 30°C for 3 h and then pulse labeled at 30°C, and chased for 0 and 1 h at 30°C. Intact and PEP4 processed bands migrate at 70 and 56 kDa, respectively. The percentage of processing after 1 h was quantified using a PhosphorImager. (B) Same as in A, except that ret1Δ1-285, sec27Δ1-285, and control (CEN::SEC27) cells were grown at 24°C, pulse labeled for 20 min at 24°C, and chased for 0 and 1 h at 24°C. Note that ret1Δ1-285 cells show a strong KKTN retrieval defect at both 24 and 30°C, whereas sec27Δ1-285 cells behave like wild-type. (C) sec27Δ1-285 and sec27-95 cells display a kinetic delay in CPY transport at permissive temperature. Analysis of CPY transport in sec27Δ1-285, sec27-95, ret1Δ1-285, ret1-1, and wild-type (WT) cells. Cells were radiolabeled for 10 min at 24°C and chased for 0, 15, and 60 min. p1, p2, and mature forms of CPY are indicated, and processing to the mature form is quantified.