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Molecular and Cellular Biology, April 2001, p. 2337-2348, Vol. 21, No. 7
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.7.2337-2348.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Protein Import Channel of the Outer Mitochondrial Membrane: a Highly Stable Tom40-Tom22 Core Structure Differentially Interacts with Preproteins, Small Tom Proteins, and Import Receptors

Chris Meisinger,1 Michael T. Ryan,1,dagger Kerstin Hill,2 Kirstin Model,1 Joo Hyun Lim,1,3 Albert Sickmann,4 Hanne Müller,1 Helmut E. Meyer,4 Richard Wagner,2 and Nikolaus Pfanner1,*

Institut für Biochemie und Molekularbiologie1 and Fakultät für Biologie,3 Universität Freiburg, D-79104 Freiburg, Biophysik, Universität Osnabrück, FB Biologie/Chemie, D-49034 Osnabrück,2 and Proteinstrukturlabor, Institut für Immunologie, Ruhr-Universität Bochum, D-44780 Bochum,4 Germany

Received 15 November 2000/Returned for modification 19 December 2000/Accepted 3 January 2001

The preprotein translocase of the yeast mitochondrial outer membrane (TOM) consists of the initial import receptors Tom70 and Tom20 and a ~400-kDa (400 K) general import pore (GIP) complex that includes the central receptor Tom22, the channel Tom40, and the three small Tom proteins Tom7, Tom6, and Tom5. We report that the GIP complex is a highly stable complex with an unusual resistance to urea and alkaline pH. Under mild conditions for mitochondrial lysis, the receptor Tom20, but not Tom70, is quantitatively associated with the GIP complex, forming a 500K to 600K TOM complex. A preprotein, stably arrested in the GIP complex, is released by urea but not high salt, indicating that ionic interactions are not essential for keeping the preprotein in the GIP complex. Under more stringent detergent conditions, however, Tom20 and all three small Tom proteins are released, while the preprotein remains in the GIP complex. Moreover, purified outer membrane vesicles devoid of translocase components of the intermembrane space and inner membrane efficiently accumulate the preprotein in the GIP complex. Together, Tom40 and Tom22 thus represent the functional core unit that stably holds accumulated preproteins. The GIP complex isolated from outer membranes exhibits characteristic TOM channel activity with two coupled conductance states, each corresponding to the activity of purified Tom40, suggesting that the complex contains two simultaneously active and coupled channel pores.

* 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.

dagger Present address: Department of Biochemistry, La Trobe University, 3086 Melbourne, Victoria, Australia.

Molecular and Cellular Biology, April 2001, p. 2337-2348, Vol. 21, No. 7
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.7.2337-2348.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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