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

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 ϳ42-kDa protein. Mammalian mitofusins Mfn1 and Mfn2 are large GTPases of the mitochondrial outer membrane that mediate mitochondrial fusion (1-3). They also contain two coiled-coil domains or heptad repeats (HR1 and HR2) 2 (3). The major portion of each of the proteins, including the N terminus-proximal GTPase domain and HR1 and the C terminus-proximal HR2, is exposed to the cytosol (4). The attachment to the outer membrane is mediated by two membrane-spanning segments that are separated by a small intermembrane space loop and that are located between the HR1 and HR2 repeats. Human Mfn1 and Mfn2 are highly homologous proteins with 62% identity at the amino acid level with both, however, being essential proteins. The homozygous Ϫ/Ϫ Mfn1-null and Ϫ/Ϫ Mfn2-null mice die during embryonic development (5). The mouse embryonic fibroblasts (MEF), derived from the Ϫ/Ϫ Mfn1-null or Ϫ/Ϫ Mfn2-null embryos, have distinct mitochondrial morphology defects, and exhibit severe reduction in mitochondrial fusion activity (5). Thus, Mfn1 and Mfn2 may have different functions in mitochondrial fusion. Unlike MEF lacking Mfn1 or Mfn2, MEF lacking both mitofusins completely lack mitochondrial fusion capacity and show severe cellular dysfunction (6). Functions of Mfn2 that are independent of its fusion activity, i.e. controlling mitochondrial metabolism and repressing vascular smooth muscle cell proliferation, have also been reported (7, 8).Mfn2 and Mfn1 have the capacity to form homo-and heterooligomers, as demonstrated by co-immunoprecipitation of tagged proteins (5, 9). At least one of the mitofusins is required on each of the adjacent mitochondria to promote mitochondrial fusion (10). The initial step of this process is characterized by tethering of two adjacent mitochondria via assembly of a mitofusin complex, which is mediated by HR2 forming a dimeric, antiparallel coiled-coil (10). The subsequent steps of mitochondrial fusion, including the exact function of the GTPase domains, are less well understood. In yeast, an interaction of the mitofusin homolog Fzo1, involving the GTPase domain and the HR domains, is essential for fusion (11). In the same organism, mitochondrial fusion has been reconstituted in vitro. Mitochondrial fusion proceeds through discreet and sequential mitochondrial outer and inner membrane fusion events, with both steps requiring GTP hydrolysis (12). A third GTPase, Opa1, appears also 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 proteolyti...

Stomatin-like Protein 2 Deficiency in T Cells Is Associated with Altered Mitochondrial Respiration and Defective CD4+ T Cell Responses

“…SLP-2 is tightly associated with the mitochondrial inner membrane (32,33), and other members of the SPFH family have been linked to the organization of various membrane microdomains (26)(27)(28)(29)(30)(31). Therefore, it is plausible to suggest that SLP-2 promotes the compartmentalization of the mitochondrial inner membrane into CL-enriched microdomains.…”

Mitochondrial dynamics and apoptosis

“…Knockdown of MIB leads to elongation of the mitochondrial network, indicating that MIB regulates mitochondrial fission (Eura et al 2006). Stomatin-like protein 2 (Stoml2) was identified in a complex with Mfn2 in healthy cells (Hajek et al 2007) and is associated with the inner mitochondrial membrane facing the inter- Mitochondrial phospholipase D (mitoPLD) is another protein implicated in mitochondrial fusion (Choi et al 2006). Loss of mitoPLD expression induced by RNA interference (RNAi) leads to fragmentation of the mitochondrial network.…”

Prohibitins and the functional compartmentalization of mitochondrial membranes

“…Similarly to prohibitins, the function of SLP-2 has recently been linked to mitochondrial fusion and the processing of OPA1. Crosslinking studies led to the identification of the GTPase mitofusin-2 (MFN2), a central component of the fusion machinery in the mitochondrial outer membrane (Hoppins et al, 2007), as an interaction partner of SLP-2 (Hajek et al, 2007). SLP-2, but not PHB2, is required for mitochondrial hyperfusion, which is induced in response to stress conditions including UV irradiation or low concentrations of cycloheximide (Tondera et al, 2009).…”