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J. Biol. Chem., Vol. 280, Issue 29, 27375-27382, July 22, 2005

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Unfolding-resistant Translocase Targeting

A NOVEL MECHANISM FOR OUTER MITOCHONDRIAL MEMBRANE LOCALIZATION EXEMPLIFIED BY THE B2 REGULATORY SUBUNIT OF PROTEIN PHOSPHATASE 2A^*

Ruben K. Dagda, Chris A. Barwacz, J. Thomas Cribbs, and Stefan Strack

From the Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242

Heterotrimeric serine/threonine protein phosphatase 2A (PP2A) consists of scaffolding (A), catalytic (C), and variable (B, B', and B'') subunits. Variable subunits dictate subcellular localization and substrate specificity of the PP2A holoenzyme. The B regulatory subunit gene is mutated in spinocerebellar ataxia type 12, and one of its splice variants, B2, targets PP2A to mitochondria to promote apoptosis in PC12 cells (Dagda, R. K., Zaucha, J. A., Wadzinski, B. E., and Strack, S. (2003) J. Biol. Chem. 278, 24976-24985). Here, we report that B2 is localized to the outer mitochondrial membrane by a novel mechanism, combining a cryptic mitochondrial import signal with a structural arrest domain. Scanning mutagenesis demonstrates that basic and hydrophobic residues mediate mitochondrial association and the proapoptotic activity of B2. When fused to green fluorescent protein, the N terminus of B2 acts as a cleavable mitochondrial import signal. Surprisingly, full-length B2 is not detectably cleaved and is retained at the outer mitochondrial membrane, even though it interacts with the TOM22 import receptor, as shown by luciferase complementation in intact cells. Mutations that open the C-terminal -propeller of B2 facilitate mitochondrial import, indicating that this rigid fold acts as a stop-transfer domain by resisting the partial unfolding step prerequisite for matrix translocation. Because hybrids of prototypical import and -propeller domains recapitulate this behavior, we predict the existence of other similarly localized proteins and a selection against highly stable protein folds in the mitochondrial matrix. This unfolding-resistant targeting to the mitochondrial translocase is necessary but not sufficient for the proapoptotic activity of B2, which also requires association with the rest of the PP2A holoenzyme.

Received for publication, April 5, 2005 , and in revised form, May 24, 2005.

^* This work was supported by National Institutes of Health Grant NS43254, American Heart Association Grant AHA0455653Z, a research grant from the United Mitochondrial Disease Foundation (to S. S.), and National Research Service Award Predoctoral Fellowship NS049659 (to R. K. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, University of Iowa Carver College of Medicine, 2-432 BSB, 51 Newton Rd., Iowa City, IA 52242. Tel.: 319-384-4439; Fax: 319-335-8930; E-mail: stefan-strack{at}uiowa.edu.

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