Fig. 4. The CID of Pcf11p is essential for cell viability. (A) pcf11-13 complements a complete chromosomal deletion of PCF11. Strain NA53 was transformed with plasmid-encoded PCF11, ΔC208 or the CID mutants pcf11-13, ΔN21-71, ΔN108, ΔN126 and ΔN266. Transformants were forced to lose the URA3-marked plasmid pFL38-PCF11 on 5-FOA plates at 23°C. (B) Mutations in pcf11-13 cause temperature sensitivity. Serial dilutions of PCF11 and pcf11-13 cells were incubated at 30 and 37°C. (C) Cross-complementation of the pcf11-2 allele in trans. The pcf11-2 strain was transformed with plasmids encoding PCF11, pcf11-2, the CID mutants pcf11-13, ΔN21-71, ΔN108, ΔN126 and ΔN266 or empty plasmid. Transformants were grown at restrictive conditions (37°C) for the pcf11-2 allele. (D) Cross-complementation of the pcf11-13 allele in trans. The pcf11-13 strain was transformed with plasmid-encoded PCF11, ΔN21-71, ΔN108, ΔN126, ΔC208, ΔC296, ΔC353 and ΔC446. Transformants were grown at restrictive growth conditions (37°C) for the pcf11-13 allele. (E) Western blot analysis of total protein extracts obtained from pcf11-13 (lanes 1–5) and pcf11-2 strains (lanes 6–9) that also carry plasmid-borne N- or C-terminally truncated deletions of Pcf11p as indicated. All deletion proteins (indicated by arrows) were N-terminal in-frame fusions with two IgG binding domains. Pcf11-13p (also containing two N-terminal IgG binding domains) and Pcf11-2 proteins are indicated by asterisks. The blot was consecutively decorated with a polyclonal anti-Pcf11p serum and anti-rabbit IgG–horseradish peroxidase conjugate.