The mitochondrial citrate synthase from Tetrahymena thermophila does not form an intermediate filament

Abstract

The mitochondrial citrate synthase (mCS) purified from the ciliate Tetrahymena thermophila has been reported to form intermediate-filament-like structures during conjugation and to self-assemble into fibers when recombinantly expressed. This would represent a rare example of a tractable and recent origin of a novel cytoskeletal element. In an attempt to investigate the evolutionary emergence of this behavior, we re-investigated the ability of Tetrahymena's mCS to form filaments in vivo. Using strep-tagged mCS in Tetrahymena and monoclonal antibodies, we found no evidence of filamentous structures during conjugation or starvation. Extensive biochemical characterization of mCS revealed that the self-assembly of recombinant protein is triggered by a specific chemical moiety shared by MES and HEPES buffers used in previous studies. The absence of indicative phenotypes in fiber-deficient GFP-tagged mutants indicates that Tetrahymena mCS did not evolve a structural role in sexual reproduction or metabolic regulation.

Keywords: Citrate synthase; Conjugation; Intermediate filament; MES buffer; Tetrahymena.