AFFINITY PURIFICATION, OVEREXPRESSION, AND CHARACTERIZATION OF CHAPERONIN-10 HOMOLOGS SYNTHESIZED WITH AND WITHOUT N-TERMINAL ACETYLATION

Utilizing the ability of bacterial chaperonin 60 (GroEL) to functional ly interact with chaperonin 10 (Cpn10) homologues in an ATP-dependent fashion, we have purified substantial amounts of mammalian, chloroplas t, and thermophilic Cpn10 homologues from their natural host. In addit ion, large amounts of recombinant rat Cpn10 were produced in Escherich ia coli and found to be identical to its authentic counterpart except for the lack of N-terminal acetylation. By comparing these two forms o f Cpn10, it was found that acetylation does not influence the oligomer ic structure of Cpn10 and is not essential for chaperone activity or m itochondrial import in vitro. In contrast, N-terminal acetylation prov ed crucial in the protection of Cpn10 against degradation by N-ethylma leimide-sensitive proteases derived from organellar preparations of ra t liver. The availability of large amounts of both affinity-purified a nd recombinant Cpn10 will facilitate not only further characterization of the eukaryotic folding machinery but also further scrutiny of the reported function of Cpn10 as early pregnancy factor.