Biochemical Study of Proteins of Cortical Cytoskeleton in the Ciliate Isotricha prostoma1

In Isotricha prostoma, proteins of isolated cortices retaining the ecto-endoplasmic fibrillar boundary (EEB) have been separated by one- and two-dimensional gel electrophoresis. In addition to that of tubulin, four main bands corresponding to polypeptide molecular weights of 185, 35, 23, and 22 kd have been observed on 9% SDS-polyacrylamide gels. Supernumerary major bands within a low molecular weight range (11-19 kd) are evident after separation on 7.5-15% polyacrylamide gradient gels. Two-dimensional analysis discloses - 150 polypeptide spots, most of them focusing within an acidic pl range 4.6-5.3. The cortical fibrillar structures are still distinguishable after incubation in solutions containing 1.0 M K. Cl (12 h) or 0.6 M Kl (8 h). A prolonged KI treatment (52 h), however, results in the removal of EEB filaments (?4 nm in diameter) and of a large part of the kinetid elements, so that the insoluble material is mainly represented by a reticulum of 6-nm filaments interconnecting the proximal regions of kinetosomes. Two proteins with MW of 58 and 63 kd appear to be constituents of this kinetosome-associated filamentous reticulum. Some of the cortical proteins with MW ≤23 kd are the most likely candidates for components of EEB filaments. The absence of decoration with heavy meromyosin confirms that actin is not a major component of Isotricha cortical cytoskeleton.