Fig. 4.

Identification of the Chr2-HA ER form and yapsin cleavage products. (A) Western blot analysis of intracellular extracts of WT cells incubated at 30°C and sec18-1 cells after shift to the restrictive temperature of 37°C. (B) Colony immunoblot analysis of Crh2-HA secretion in wt and sec18-1 cells incubated at 30°C and processed as described in the legend of Fig. 1C. (C) wt and yps1Δ yps2Δ cells harboring CRH2-HA were grown to logarithmic phase in synthetic medium. The cells were harvested, and total cell extracts (C) were prepared. Medium proteins (M) were precipitated with trichloroacetic acid-deoxycholate. Samples were analyzed by Western blotting as described in Materials and Methods. Medium proteins corresponding to equivalent numbers of cells were loaded into each lane. Cellular proteins were normalized by protein concentration. (D) Schematic representation of Crh2-HA with the sequences of Yps1/2p cleavage sites that are consistent with the 40- to 50-kDa Western blot band are indicated. The arrows indicate the residue where cleavage is predicted.