Figure 9.

Human PERLD1 is a functional homologue of Per1p, and H177 and H326 are required for Per1p activity. (A) Alignment of the amino acid sequences of Per1p homologues. Amino acid sequences of Homo sapiens (GenBank Accession No. AAH10652), Mus musculus (Accession No. NP_001028709), Schizosaccharomyces pombe (Accession No. CAB90152), and S. cerevisiae (Accession No. NP_009973) proteins were aligned using ClustalW. (B) Spot test of CFW and temperature sensitivities in per1 mutant cells. Top panels, isogenic per1Δ cells carrying the pRS316T (vector), pMF917 (PER1), or pMF942 (Human PERLD1) were spotted on YPAD or YPAD plates containing 7.5 μg/ml CFW after making a 5× serially diluted solution and then incubated for 2 d at 30 or 37°C. Bottom panels, isogenic per1Δ cells carrying the pRS316T (vector), pMF918 (PER1-HA), pMF931 (S19A), pMF932 (H102A), pMF933 (K104A), pMF934 (S118A), pMF935 (S122A), pMF936 (S173A), pMF937 (H177A), pMF939 (D315A), or pMF940 (H326A) were spotted on YPAD or YPAD plates containing 7.5 μg/ml CFW and then incubated for 2 d at 30 or 37°C. (C) Human PERLD1 expression restores the amount of Gas1p to per1Δ cells. Isogenic per1Δ cells carrying pRS316T, pMF917, or pMF942 were grown overnight to midlog phase. Equal cell numbers were lysed, and total protein extracts were analyzed by immunoblotting using anti-Gas1p and anti-Dpm1p. (D) H177 and H326 are essential for Per1p activity. Isogenic per1Δ cells carrying the pRS316T, pMF918, pMF931, pMF932, pMF933, pMF934, pMF935, pMF936, pMF937, pMF939, or pMF940 were grown overnight to midlog phase. Equal cell numbers were lysed, and total protein extracts were analyzed by immunoblotting using anti-Gas1p, anti-HA, and anti-Dpm1p.