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Molecular and Cellular Biology, August 2000, p. 5879-5887, Vol. 20, No. 16
Institut für Biochemie und
Molekularbiologie1 and Fakultät
für Biologie,2 Universität Freiburg,
D-79104 Freiburg, Institut für Mikrobiologie,
Universität Hohenheim, D-70593 Stuttgart,3
and Abteilung für Zellbiochemie, Medizinische
Fakultät der Ruhr-Universität Bochum, D-44780
Bochum,4 Germany
Received 3 February 2000/Returned for modification 27 March
2000/Accepted 23 May 2000
The import motor for preproteins that are targeted into the
mitochondrial matrix consists of the matrix heat shock protein Hsp70
(mtHsp70) and the translocase subunit Tim44 of the inner membrane.
mtHsp70 interacts with Tim44 in an ATP-dependent reaction cycle, binds
to preproteins in transit, and drives their translocation into the
matrix. While different functional mechanisms are discussed for the
mtHsp70-Tim44 machinery, little is known about the actual mode of
interaction of both proteins. Here, we have addressed which of the
three Hsp70 regions, the ATPase domain, the peptide binding domain, or
the carboxy-terminal segment, are required for the interaction with
Tim44. By two independent means, a two-hybrid system and
coprecipitation of mtHsp70 constructs imported into mitochondria, we
show that the ATPase domain interacts with Tim44, although with a
reduced efficiency compared to the full-length mtHsp70. The interaction
of the ATPase domain with Tim44 is ATP sensitive. The peptide binding
domain and carboxy-terminal segment are unable to bind to Tim44 in the
absence of the ATPase domain, but both regions enhance the interaction
with Tim44 in the presence of the ATPase domain. We conclude that the
ATPase domain of mtHsp70 is essential for and directly interacts with
Tim44, clearly separating the mtHsp70-Tim44 interaction from the
mtHsp70-substrate interaction.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mitochondrial Protein Import Motor: the ATPase Domain of Matrix
Hsp70 Is Crucial for Binding to Tim44, while the Peptide Binding Domain
and the Carboxy-Terminal Segment Play a Stimulatory Role
*
Corresponding author. Mailing address: Institut
für Biochemie und Molekularbiologie, Universität Freiburg,
Hermann-Herder-Straße 7, D-79104 Freiburg, Germany. Phone: 49-761 203 5224. Fax: 49-761 203 5261. E-mail: pfanner{at}uni-freiburg.de.
Molecular and Cellular Biology, August 2000, p. 5879-5887, Vol. 20, No. 16
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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