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

doi:10.1128/MCB.21.7.2337-2348.2001

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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^, 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 Tom70and Tom20 and a ~400-kDa (400 K) general import pore (GIP) complexthat includes the central receptor Tom22, the channel Tom40, andthe three small Tom proteins Tom7, Tom6, and Tom5. We report thatthe GIP complex is a highly stable complex with an unusual resistanceto urea and alkaline pH. Under mild conditions for mitochondriallysis, the receptor Tom20, but not Tom70, is quantitatively associatedwith the GIP complex, forming a 500K to 600K TOM complex. A preprotein,stably arrested in the GIP complex, is released by urea but nothigh salt, indicating that ionic interactions are not essentialfor keeping the preprotein in the GIP complex. Under more stringentdetergent conditions, however, Tom20 and all three small Tom proteinsare released, while the preprotein remains in the GIP complex.Moreover, purified outer membrane vesicles devoid of translocasecomponents of the intermembrane space and inner membrane efficientlyaccumulate the preprotein in the GIP complex. Together, Tom40and Tom22 thus represent the functional core unit that stablyholds accumulated preproteins. The GIP complex isolated from outermembranes exhibits characteristic TOM channel activity with twocoupled conductance states, each corresponding to the activityof purified Tom40, suggesting that the complex contains two simultaneouslyactive and coupled channelpores.

^* Corresponding author. Mailing address: Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Straße7, D-79104 Freiburg, Germany. Phone: 49-761 203 5224. Fax: 49-761203 5261. E-mail: pfanner{at}uni-freiburg.de.

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

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