Coordinate synthesis but discrete localization of homologous N-glycosylated proteins, CLP and CLB, in Naegleria pringsheimi flagellates

Abstract

The synchronous amoebae-to-flagellates differentiation of Naegleria pringsheimi has been used as a model system to study the formation of eukaryotic flagella. We cloned two novel genes, Clp, Class I on plasma membrane and Clb, Class I at basal bodies, which are transiently expressed during differentiation and characterized their respective protein products. CLP (2,087 amino acids) and CLB (1,952 amino acids) have 82.9% identity in their amino acid sequences and are heavily N-glycosylated, leading to an ∼ 100 × 103 increase in the relative molecular mass of the native proteins. In spite of these similarities, CLP and CLB were localized to distinct regions: CLP was present on the outer surface of the plasma membrane, whereas CLB was concentrated at a site where the basal bodies are assembled and remained associated with the basal bodies. Oryzalin, a microtubule toxin, inhibited the appearance of CLP on the plasma membrane, but had no effect on the concentration of CLB at its target site. These data suggest that N. pringsheimi uses separate mechanisms to transport CLP and CLB to the plasma membrane and to the site of basal body assembly, respectively. © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.