Volume 270,
Number 37,
Issue of September 15, pp. 22037-22043, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Affinity
Purification, Overexpression, and Characterization of Chaperonin 10
Homologues Synthesized with and without N-terminal Acetylation
(Received for publication, May 24, 1995; and in revised form, June 23, 1995)
Michael T.
Ryan
, <WBR>
Dean J.
Naylor
, <WBR>, <WBR>
Nicholas J.
Hoogenraad
, <WBR>
Peter B.
Høj
Utilizing the ability of bacterial chaperonin 60 (GroEL) to
functionally interact with chaperonin 10 (Cpn10) homologues in an
ATP-dependent fashion, we have purified substantial amounts of
mammalian, chloroplast, and thermophilic Cpn10 homologues from their
natural host. In addition, large amounts of recombinant rat Cpn10 were
produced in Escherichia coli and found to be identical to its
authentic counterpart except for the lack of N-terminal acetylation. By
comparing these two forms of Cpn10, it was found that acetylation does
not influence the oligomeric structure of Cpn10 and is not essential
for chaperone activity or mitochondrial import in vitro. In
contrast, N-terminal acetylation proved crucial in the protection of
Cpn10 against degradation by N-ethylmaleimide-sensitive
proteases derived from organellar preparations of rat liver. The
availability of large amounts of both affinity-purified and 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.
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