Heterologous expression and folding analysis of a β-tubulin isotype from the Antarctic ciliate Euplotes focardii

Abstract

Mammalian tubulins and actins attain their native conformation following interactions with CCT (the cytosolic chaperonin containing t-complex polypeptide 1). To study the β-tubulin folding in lower eukaryotes, an isotype of β-tubulin (β-T1) from the Antarctic ciliate Euplotes focardii, was expressed in Escherichia coli. Folding analysis was performed by incubation of the 35S-labeled, denatured β-T1 in the presence, or absence, of purified rabbit CCT and cofactor A, a polypeptide that stabilizes folded monomeric β-tubulin. We show for the first time in protozoa that β-tubulin folding is assisted by CCT and requires cofactor A. In addition, we observed that E. focardii β-T1 competes with human β5 tubulin isotype for binding to CCT. The affinity of CCT to E. focardii β-T1 and β5 tubulin are compared. Finally, the mitochondrial chaperonin mt-cpn60 binds to β-T1 but is unable to release it in a native or quasi-native state.