Functional analysis of domain-mutated Vps27 derivatives. (A) Schematic drawing of Vps27 intramolecular domains. The binding partner of each domain is also shown. CB, clathrin-binding motif; PTAPL, PTAP-like amino-acid sequence. (B) Immunoblot analysis of Vph1-EGFP in vps27 mutants. The data are presented as in Fig. 1 A. In the VPS27-deleted (vps27Δ) strain, normal (WT) or mutant forms of Vps27 were expressed. The designation of mut indicates introduction of point mutations in the denoted domains. The cleavage rate for each variant [(band intensity of CL)/(band intensities of FL + CL) × 100 (%)] was calculated from three independent experiments (right). The error bars indicate standard deviation. (C) Immunoblot analysis of yEGFP-Pho8 expressed in vps27 mutant strains. The asterisk indicates a nonspecific band. (D) Immunoblot analysis of Vph1-EGFP expressed in chc1 (clathrin heavy chain) mutant or WT strain cultured at 23°C for 24 h (23°C) or cultured at 23°C for 16 h and subsequently cultured at 31°C for 8 h (31°C). The cleavage rates calculated as in B from three independent experiments are also shown. Error bars indicate standard deviation.