Figure 5.
Pcs1 and Spc25 proteins share sequence similarity. (A) Sequence alignment of the Spindle_Spc25 domain of Pcs1 and Spc25 proteins. Mutated Pcs1 residues described in the (B) are indicated. A selected set of Pcs1 and Spc25 protein family sequences, covering a coiled coil region (the first helix shown) and the globular sequence domain, were aligned with T-coffee (default parameters, reviewed in ref. 70). The sequences used are derived from the NCBI RefSeq database71 with the following accessions for the Pcs1 sequence family: S.cer. (Saccharomyces cerevisiae, NP_010009.1), C.alb. (Candida albicans, XP_719422.1), A.nid. (Aspergillus nidulans, XP_664652.1), G.zea. (Gibberella zeae, XP_382110.1), P.chr. (Penicillium chrysogenum, XP_002559041.1), S.pom. (Schizosaccharomyces pombe, NP_001018298.1), C.neo. (Cryptococcus neoformans, XP_571257.1), C.cin. (Coprinopsis cinerea, XP_001833086.1), E.cun. (Encephalitozoon cuniculi, NP_585824.1), N.vec. (Nematostella vectensis, XP_001632729.1); the Spc25 protein family was represented by S.cer. (NP_010934.1), S.pom. (NP_588208.1), C.neo. (XP_570912.1), E.cun. (NP_586429.1), N.vec. (XP_001635865.1), S.man. (Schistosoma mansoni, XP_002580161.1), I.sca. (Ixodes scapularis, XP_002434782.1), S.pur. (Strongylocentrotus purpuratus, XP_797276.1), B.flo. (Branchiostoma floridae, XP_002592012.1), H.sap. (Homo sapiens, NP_065726.1), A.tha. (Arabidopsis thaliana, NP_566900.1), C.rei. (Chlamydomonas reinhardtii, XP_001697914.1). Stretches of unaligned sequence have been removed and the number of deleted residues is indicated in brackets. The top two lines of the alignment indicate predicted secondary structure and corresponding confidence values of S. pombe Pcs1 calculated with PSIPRED.72 The bottom line shows the secondary structure of S. cerevisiae Spc25 derived from X-ray crystallography (2FTX) as depicted from Wei et al. 2006.55 (B) Serial dilutions of wild-type cells (wt) (JG15414), pcs1Δ mutant cells (JG14821) and cells carrying the indicated pcs1 mutations pcs1(L140A) (JG15549), pcs1(F154A) (JG15550), pcs1(F154D) (JG15551), pcs1(F212A) (JG15552), pcs1(F212D) (JG15553) were spotted on YES medium or YES medium containing 15 mg/ml of thiabenda-zole (TBZ) and grown for 2 days at 32°C.