Heterologous expression and folding analysis of a beta-tubulin isotype from the Antarctic ciliate Euplotes focardii.
Mammalian tubulins and actins attain their native conformation following interactions with CCT (the cytosolic chaperonin containing t-complex polypeptide 1). To study the beta-tubulin folding in lower eukaryotes, an isotype of beta-tubulin (beta-T1) from the Antarctic ciliate Euplotes focardii, was expressed in Escherichia coli. Folding analysis was performed by incubation of the 35S-labeled, denatured beta-T1 in the presence, or absence, of purified rabbit CCT and cofactor A, a polypeptide that stabilizes folded monomeric beta-tubulin. We show for the first time in protozoa that beta-tubulin folding is assisted by CCT and requires cofactor A. In addition, we observed that E. focardiibeta-T1 competes with human beta5 tubulin isotype for binding to CCT. The affinity of CCT to E. focardiibeta-T1 and beta5 tubulin are compared. Finally, the mitochondrial chaperonin mt-cpn60 binds to beta-T1 but is unable to release it in a native or quasi-native state.
Pucciarelli
Sandra
S
Dipartimento di Biologia Molecolare, Cellulare e Animale, Università di Camerino, Camerino, Italy. sandra.pucciarelli@unicam.it
Miceli
Cristina
C
Melki
Ronald
R
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D018834:Chaperonin 60 / Q000378:metabolism; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D016812:Euplotes / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014404:Tubulin / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism
D018834:Chaperonin 60; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D014404:Tubulin; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins