Open AccessHeterologous expression and folding analysis of a β‐tubulin isotype from the Antarctic ciliate Euplotes focardii
Author(s) -
Pucciarelli Sandra,
Miceli Cristina,
Melki Ronald
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2002.03346.x
Subject(s) - tubulin , chaperonin , ciliate , isotype , biology , chaperone (clinical) , biochemistry , protein folding , microbiology and biotechnology , native state , microtubule , genetics , antibody , medicine , pathology , monoclonal antibody
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 35 S‐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.