Identification and molecular characterization of a putative ciliary WD-repeat protein in the ciliate Euplotes octocarinatus

• Published in: European journal of protistology 2000-07, Vol.36 (2), p.127-137
• Main Author: Pluemper, E
• Format: Article
• Language: English
• Subjects: Cilia; CTXP80 protein; double prime WD repeat proteins; Euplotes octocarinatus; Freshwater; Genetic code; Hypotrichous ciliate; Transducin; WD repeat proteins
• ISSN: 0932-4739; 1618-0429
• DOI: 10.1016/S0932-4739(00)80030-9

Cilia of the hypotrichous ciliate Euplotes octocarinatus were subjected to Triton X-114 fractionation followed by two-dimensional electrophoresis. CTXP80, an 80 kDa protein with a pI of 6.5 was identified as the most dominant protein in the detergent phase, which is enriched in hydrophobic, membrane associated proteins. After in-gel digestion with trypsin, two fragments of CTXP80 were microsequenced and the information obtained was used to generate degenerate primers to amplify parts of the corresponding macronuclear gene. Successive amplification of the entire CTXP80 encoding macronuclear chromosome and sequence analysis revealed an open reading frame of 2,151 bp. The open reading frame codes for a protein with an estimated Mr of 77,300 and an estimated pI of 6.12 and contains three in-frame TGA codons coding for cysteine. TAG is used as the termination codon. Fractionation of CTXP80 into the detergent phase as well as a putative myristylation site at the N-terminus of the protein suggest that CTXP80 is membrane associated facing the ciliary matrix. A total of seven putative WD-repeats were identified within the open reading frame which makes CTXP80 a novel member of the WD-repeat family of proteins.