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 approximately 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.
Highlights
Domain and HR1 and the C terminus-proximal HR2, is exposed to the cytosol [4]
A third GTPase, Opa1, appears to be involved in the process of mitochondrial fusion [13]. This GTPase is synthesized as a precursor, which is processed by a matrix-processing peptidase to a mature form, large Opa1 (l-Opa1). l-Opa1 is anchored to the inner mitochondrial membrane and can undergo another proteolytic processing, which cleaves off the transmembrane segment and forms the small form (s-Opa1) [14]
Our results suggest that stomatin-like protein 2 (Stoml2) is a novel mitochondrial intermembrane space/inner membrane-localized protein that forms a large hetero-oligomeric complex with mitofusin 2 (Mfn2)
Summary
Plasmid Construction and in Vitro Transcription/ Translation—To construct a plasmid encoding a His-tagged protein corresponding to amino acids 1– 405 of Mfn (HisMfn2-(1– 405)), a 1215-nucleotide-long fragment of an MFN2 cDNA was amplified by PCR from pBSKIAA0214, which carries a human heart cDNA of MFN2 [21], and cloned into the NdeI-BamHI sites of pET-15b. Sample Preparation and Western Blot Analysis—To remove contaminating rabbit IgGs present in the rabbit reticulocyte lysate-based TNT T7-coupled transcription/translation system, which could interfere, in the Western blot analysis, with detection of in vitro synthesized Stoml, samples of in vitro synthesized Stoml were incubated with protein A-Sepharose beads in an appropriate buffer (50 mM NaCl, 0.1 mM EDTA, 0.1% Tween 20, 25 mM Tris-HCl, pH 7.5 (25 °C)) for 1 h at 4 °C, and the Sepharose beads were removed by centrifugation. The FA-cross-linked cells or FA-cross-linked purified mitochondria were incubated with buffer containing 2% SDS, 100 mM NaCl, and 10 mM Tris-HCl, pH 8.0 (25 °C) for 15 min and centrifuged to remove insoluble debris, and the resulting extracts were used for Western blot analysis. After four washes in PBS, the coverslips were mounted onto microscope slides in FluoroGuard antifade reagent (Bio-Rad) and analyzed on a Zeiss 410 laser-scanning microscope equipped with a 488-nm argon and a 543-nm helium neon laser, a Zeiss 63ϫ/1.25 oil objective, and Zeiss LSM software
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
