Communication of mitochondria with the rest of the cell requires beta-barrel proteins of the outer membrane. All beta-barrel proteins are synthesized as precursors in the cytosol and imported into mitochondria by the general translocase TOM and the sorting machinery SAM. The SAM complex contains two proteins essential for cell viability, the channel-forming Sam50 and Sam35. We have identified the sorting signal of mitochondrial beta-barrel proteins that is universal in all eukaryotic kingdoms. The beta-signal initiates precursor insertion into a hydrophilic, proteinaceous membrane environment by forming a ternary complex with Sam35 and Sam50. Sam35 recognizes the beta-signal, inducing a major conductance increase of the Sam50 channel. Subsequent precursor release from SAM is coupled to integration into the lipid phase. We propose that a two-stage mechanism of signal-driven insertion into a membrane protein complex and subsequent integration into the lipid phase may represent a general mechanism for biogenesis of beta-barrel proteins.

The dynamic network of mitochondria fragments under stress allowing the segregation of damaged mitochondria and, in case of persistent damage, their selective removal by mitophagy. Mitochondrial fragmentation upon depolarisation of mitochondria is brought about by the degradation of central components of the mitochondrial fusion machinery. The OMA1 peptidase mediates the degradation of long isoforms of the dynamin-like GTPase OPA1 in the inner membrane. Here, we demonstrate that OMA1-mediated degradation of OPA1 is a general cellular stress response. OMA1 is constitutively active but displays strongly enhanced activity in response to various stress insults. We identify an amino terminal stress-sensor domain of OMA1, which is only present in homologues of higher eukaryotes and which modulates OMA1 proteolysis and activation. OMA1 activation is associated with its autocatalyic degradation, which initiates from both termini of OMA1 and results in complete OMA1 turnover. Autocatalytic proteolysis of OMA1 ensures the reversibility of the response and allows OPA1-mediated mitochondrial fusion to resume upon alleviation of stress. This differentiated stress response maintains the functional integrity of mitochondria and contributes to cell survival.

Mitochondria undergo balanced fission and fusion events that enable their appropriate networking within the cell. In yeast, three factors have been identified that co-ordinate fission events at the mitochondrial outer membrane. Fis1p acts as the outer membrane receptor for recruitment of the dynamin member, Dnm1p and the WD40-repeat-containing protein Mdv1p. In mammals, the Dnm1p counterpart Drp1 has been characterized, but other components have not. Here, we report the characterization of human Fis1 (hFis1). hFis1 is inserted into the mitochondrial outer membrane via a C-terminal transmembrane domain that, along with a short basic segment, is essential for its targeting. Although expression of hFis1 does not complement the phenotype of yeast cells lacking Fis1p, overexpression of hFis1 in tissue culture cells nevertheless causes mitochondrial fragmentation and aggregation. This aggregation could be suppressed by expressing a dominant-negative Drp1 mutant (Drp1K38A). Knockdown of hFis1 in COS-7 cells using RNA interference results in mitochondrial morphology defects with notable extensions in the length of mitochondrial tubules. These results indicate that the levels of hFis1 at the mitochondrial surface influences mitochondrial fission events and hence overall mitochondrial morphology within the cell.

Mitochondria import a large number of nuclear-encoded proteins via membrane-bound transport machineries; however, little is known about regulation of the preprotein translocases. We report that the main protein entry gate of mitochondria, the translocase of the outer membrane (TOM complex), is phosphorylated by cytosolic kinases-in particular, casein kinase 2 (CK2) and protein kinase A (PKA). CK2 promotes biogenesis of the TOM complex by phosphorylation of two key components, the receptor Tom22 and the import protein Mim1, which in turn are required for import of further Tom proteins. Inactivation of CK2 decreases the levels of the TOM complex and thus mitochondrial protein import. PKA phosphorylates Tom70 under nonrespiring conditions, thereby inhibiting its receptor activity and the import of mitochondrial metabolite carriers. We conclude that cytosolic kinases exert stimulatory and inhibitory effects on biogenesis and function of the TOM complex and thus regulate protein import into mitochondria.

Cytosolic dynamin-related protein 1 (Drp1, also known as DNM1L) is required for both mitochondrial and peroxisomal fission. Drp1-dependent division of these organelles is facilitated by a number of adaptor proteins at mitochondrial and peroxisomal surfaces. To investigate the interplay of these adaptor proteins, we used geneediting technology to create a suite of cell lines lacking the adaptors MiD49 (also known as MIEF2), MiD51 (also known as MIEF1), Mff and Fis1. Increased mitochondrial connectivity was observed following loss of individual adaptors, and this was further enhanced following the combined loss of MiD51 and Mff. Moreover, loss of adaptors also conferred increased resistance of cells to intrinsic apoptotic stimuli, with MiD49 and MiD51 showing the more prominent role. Using a proximity-based biotin labeling approach, we found close associations between MiD51, Mff and Drp1, but not Fis1. Furthermore, we found that MiD51 can suppress Mff-dependent enhancement of Drp1 GTPase activity. Our data indicates that Mff and MiD51 regulate Drp1 in specific ways to promote mitochondrial fission.

Background: Various receptor proteins recruit Drp1 to drive fission of mitochondria and peroxisomes. Results: MiD49 and MiD51 recruit Drp1 specifically to mitochondria independently of receptors Fis1 and Mff. Conclusion: MiD49 and MiD51 appear to be specific to the mitochondrial fission apparatus of mammalian cells. Significance: Mitochondrial and peroxisomal fission processes can be differentially regulated.

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The intermembrane space of mitochondria contains the specific mitochondrial intermembrane space assembly (MIA) machinery that operates in the biogenesis pathway of precursor proteins destined to this compartment. The Mia40 component of the MIA pathway functions as a receptor and binds incoming precursors, forming an essential early intermediate in the biogenesis of intermembrane space proteins. The elements that are crucial for the association of the intermembrane space precursors with Mia40 have not been determined. In this study, we found that a region within the Tim9 and Tim10 precursors, consisting of only nine amino acid residues, functions as a signal for the engagement of substrate proteins with the Mia40 receptor. Furthermore, the signal contains sufficient information to facilitate the transfer of proteins across the outer membrane to the intermembrane space. Thus, here we have identified the mitochondrial intermembrane space sorting signal required for delivery of proteins to the mitochondrial intermembrane space. INTRODUCTIONMitochondria pose a great challenge for the proper delivery of proteins because of their complex architecture. Mitochondrial precursors must find their way to one of the four mitochondrial subcompartments: the outer membrane, intermembrane space, inner membrane, or matrix. As a direct consequence of this complexity, several machineries for the translocation and sorting of mitochondrial precursors have evolved. Interplay between these machineries and specific signals present in the precursors drive different protein targeting pathways (Schatz and Dobberstein, 1996;Emanuelsson and von Heijne, 2001;Jensen and Johnson, 2001;Endo et al., 2003;Koehler, 2004;Oka and Mihara, 2005;Dolezal et al., 2006;Neupert and Herrmann, 2007;Bolender et al., 2008). Initially, mitochondrial precursors are recognized in a signal-dependent manner by specific receptors and are transferred across the barrier of outer mitochondrial membrane by using the translocase of the outer membrane (TOM) complex. On the trans-side of the outer mitochondrial membrane, sorting machineries decode specific signals in precursors, and this results in the branching of protein import pathways. The most well characterized is the presequence pathway across the inner membrane driven by a cleavable and positively charged signal sequence, called a presequence, and the translocase of the inner membrane (TIM) 23 complex Endo et al., 2003;Oka and Mihara, 2005;Neupert and Herrmann, 2007;Bolender et al., 2008). The presequence is cleaved off by a specific protease liberating the mature protein. However, other mitochondrial signals are not proteolytically removed and remain as part of the native mitochondrial protein. One example is the recently identified ␤-signal that is recognized by the sorting and assembly machinery (SAM) complex to sort ␤-barrel proteins to the mitochondrial outer membrane . In other mitochondrial membrane proteins, the membrane domains, anchors, and their surrounding regions are used to some extent for selection of ...