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Identification of a Novel Mitochondrial Complex Containing Mitofusin 2 and Stomatin-like Protein 2

Hájek, Petr and Chomyn, Anne and Attardi, Giuseppe (2007) Identification of a Novel Mitochondrial Complex Containing Mitofusin 2 and Stomatin-like Protein 2. Journal of Biological Chemistry, 282 (8). pp. 5670-5681. ISSN 0021-9258. https://resolver.caltech.edu/CaltechAUTHORS:HAJjbc07

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Abstract

A reverse genetics approach was utilized to discover new proteins that interact with the mitochondrial fusion mediator mitofusin 2 (Mfn2) and that may participate in mitochondrial fusion. In particular, in vivo formaldehyde cross-linking of whole HeLa cells and immunoprecipitation with purified Mfn2 antibodies of SDS cell lysates were used to detect an ~42-kDa protein. This protein was identified by liquid chromatography and tandem mass spectrometry as stomatin-like protein 2 (Stoml2), previously described as a peripheral plasma membrane protein of unknown function associated with the cytoskeleton of erythrocytes (Wang, Y., and Morrow, J. S. (2000) J. Biol. Chem. 275, 8062–8071). Immunoblot analysis with anti-Stoml2 antibodies showed that Stoml2 could be immunoprecipitated specifically with Mfn2 antibody either from formaldehyde-cross-linked and SDS-lysed cells or from cells lysed with digitonin. Subsequent immunocytochemistry and cell fractionation experiments fully supported the conclusion that Stoml2 is indeed a mitochondrial protein. Furthermore, demonstration of mitochondrial membrane potential-dependent import of Stoml2 accompanied by proteolytic processing, together with the results of sublocalization experiments, suggested that Stoml2 is associated with the inner mitochondrial membrane and faces the intermembrane space. Notably, formaldehyde cross-linking revealed a "ladder" of high molecular weight protein species, indicating the presence of high molecular weight Stoml2-Mfn2 hetero-oligomers. Knockdown of Stoml2 by the short interfering RNA approach showed a reduction of the mitochondrial membrane potential, without, however, any obvious changes in mitochondrial morphology.


Item Type:Article
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https://doi.org/10.1074/jbc.M608168200DOIUNSPECIFIED
Additional Information:© 2007 the American Society for Biochemistry and Molecular Biology. Received for publication, August 25, 2006 , and in revised form, October 27, 2006. Originally published In Press as doi:10.1074/jbc.M608168200 on November 22, 2006. We are very grateful to Jaehyoung Cho for his excellent suggestions and for fruitful discussions and to Heenam Park and Rosario Zedan for technical assistance. We also thank Ansgar Santel for providing anti-Mfn1 antibodies. This work was supported by Public Health Service Grant GM11726 (to G. A.). 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.
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Funding AgencyGrant Number
NIHGM11726
Issue or Number:8
Record Number:CaltechAUTHORS:HAJjbc07
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:HAJjbc07
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7604
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:12 Mar 2007
Last Modified:02 Oct 2019 23:43

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