Chapter 48 Isolation of Flagellar Paraxonemal Rod Proteins

Abstract

Publisher Summary The paraxonemal rod (PR) is a well-ordered lattice of cytoskeletal filaments with properties distinct from those of microtubules, microfilaments, and intermediate filaments. It is a major component of some lower eukaryotic flagella. Paracrystalline PR of Trypanosoma is composed of two major helix-like proteins, which are present in approximately equal molar ratios and are resistant to extraction by nonionic detergent and low salt; in contrast, the major proteins of the coiled-sheet PR found in the Euglena emergent flagellum exhibit a greater degree of heterogeneity. PR is directly coupled to both the tip-assembled axonemal doublets and to the base-assembled surface mastigonemes. 6–12 isoelectric isoforms migrating in the range 64–75 kDa are recognized by an anti-PR monoclonal antibody, and biochemical complexity is further suggested from the heterogeneous population of cDNA clones. In addition, Euglena PR proteins are readily solubilized with detergents, salts, or chaotropes and are extremely sensitive to trypsin and Pronase digestion. Limited protease digestion of Trypanosoma PR proteins, on the other hand, consistently produces a 65-kDa protease-resistant core. The more stable PR proteins from Trypanosoma cruzi have been successfully urea-extracted and purified by column chromatography. For Euglena , thermal fractionation protocols have been developed that can produce PR-enriched flagellar fractions for in vitro flagellar reassembly studies and PR protein binding assays. Procedures for the thermal enrichment of Euglena PR proteins and purification of Trypanosoma PR proteins are summarized in this chapter. The thermal fractionation of Euglena paraxonemal rod proteins—enrichment of paraxonemal rod proteins, fractionation of PR1 and PR2, and purification of Trypanosoma paraxonemal rod proteins—are also discussed.