Prohibitins comprise an evolutionarily conserved and ubiquitously expressed family of membrane proteins with poorly described functions. Large assemblies of PHB1 and PHB2 subunits are localized in the inner membrane of mitochondria, but various roles in other cellular compartments have also been proposed for both proteins. Here, we used conditional gene targeting of murine Phb2 to define cellular activities of prohibitins. Our experiments restrict the function of prohibitins to mitochondria and identify the processing of the dynamin-like GTPase OPA1, an essential component of the mitochondrial fusion machinery, as the central cellular process controlled by prohibitins. Deletion of Phb2 leads to the selective loss of long isoforms of OPA1. This results in an aberrant cristae morphogenesis and an impaired cellular proliferation and resistance toward apoptosis. Expression of a long OPA1 isoform in PHB2-deficient cells suppresses these defects, identifying impaired OPA1 processing as the primary cellular defect in the absence of prohibitins. Our results therefore assign an essential function for the formation of mitochondrial cristae to prohibitins and suggest a coupling of cell proliferation to mitochondrial morphogenesis.

Dysfunction of mitochondria has severe cellular consequences and is linked to ageing and neurodegeneration in human. Several surveillance strategies have evolved that limit mitochondrial damage and ensure cellular integrity. Intraorganellar proteases conduct protein quality control and exert regulatory functions, membrane fusion and fission allow mitochondrial content mixing within a cell, and the autophagic degradation of severely damaged mitochondria protects against apoptosis. Here, we will summarize the current knowledge on these surveillance strategies and their role in human disease.

SummaryThe suppressor mutation, named sfhC21, that allows Escherichia coli ftsH null mutant cells to survive was found to be an allele of fabZ encoding R-3-hydroxyacyl-ACP dehydrase, involved in a key step of fatty acid biosynthesis, and appears to upregulate the dehydrase. The ftsH1(Ts) mutation increased the amount of lipopolysaccharide at 42ЊC. This was accompanied by a dramatic increase in the amount of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase [the lpxC (envA) gene product] involved in the committed step of lipid A biosynthesis. Pulse-chase experiments and in vitro assays with purified components showed that FtsH, the AAA-type membrane-bound metalloprotease, degrades the deacetylase. Genetic evidence also indicated that the FtsH protease activity for the deacetylase might be affected when acyl-ACP pools were altered. The biosynthesis of phospholipids and the lipid A moiety of lipopolysaccharide, both of which derive their fatty acyl chains from the same R-3-hydroxyacyl-ACP pool, is regulated by FtsH.

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 are dynamic organelles whose function depends on intramitochondrial phospholipid synthesis and the supply of membrane lipids from the endoplasmic reticulum. How phospholipids are transported to and in-between mitochondrial membranes remained unclear. We identified Ups1, a yeast member of a conserved family of intermembrane space proteins, as a lipid transfer protein that can shuttle phosphatidic acid between mitochondrial membranes. Lipid transfer required the dynamic assembly of Ups1 with Mdm35 and allowed conversion of phosphatidic acid to cardiolipin in the inner membrane. High cardiolipin concentrations prevented membrane dissociation of Ups1, leading to its proteolysis and inhibiting transport of phosphatidic acid and cardiolipin synthesis. Thus, intramitochondrial lipid trafficking may involve a regulatory feedback mechanism that limits the accumulation of cardiolipin in mitochondria.

Escherichia coliFtsH is an ATP-dependent protease that belongs to the AAA protein family. The second region of homology (SRH) is a highly conserved motif among AAA family members and distinguishes these proteins in part from the wider family of Walker-type ATPases. Despite its conservation across the AAA family of proteins, very little is known concerning the function of the SRH. To address this question, we introduced point mutations systematically into the SRH of FtsH and studied the activities of the mutant proteins. Highly conserved amino acid residues within the SRH were found to be critical for the function of FtsH, with mutations at these positions leading to decreased or abolished ATPase activity. The effects of the mutations on the protease activity of FtsH correlated strikingly with their effects on the ATPase activity. The ATPase-deficient SRH mutants underwent an ATP-induced conformational change similar to wild type FtsH, suggesting an important role for the SRH in ATP hydrolysis but not ATP binding. Analysis of the data in the light of the crystal structure of the hexamerization domain of Nethylmaleimide-sensitive fusion protein suggests a plausible mechanism of ATP hydrolysis by the AAA ATPases, which invokes an intermolecular catalytic role for the SRH.

Prohibitins comprise a remarkably conserved protein family in eukaryotic cells with proposed functions in cell cycle progression, senescence, apoptosis, and the regulation of mitochondrial activities. Two prohibitin homologues, Phb1 and Phb2, assemble into a high molecular weight complex of approximately 1.2 MDa in the mitochondrial inner membrane, but a nuclear localization of Phb1 and Phb2 also has been reported. Here, we have analyzed the biogenesis and structure of the prohibitin complex in Saccharomyces cerevisiae. Both Phb1 and Phb2 subunits are targeted to mitochondria by unconventional noncleavable targeting sequences at their amino terminal end. Membrane insertion involves binding of newly imported Phb1 to Tim8/13 complexes in the intermembrane space and is mediated by the TIM23-translocase. Assembly occurs via intermediate-sized complexes of approximately 120 kDa containing both Phb1 and Phb2. Conserved carboxy-terminal coiled-coil regions in both subunits mediate the formation of large assemblies in the inner membrane. Single particle electron microscopy of purified prohibitin complexes identifies diverse ring-shaped structures with outer dimensions of approximately 270 x 200 angstroms. Implications of these findings for proposed cellular activities of prohibitins are discussed.

A decline in mitochondrial activity has been associated with aging and is a hallmark of many neurological diseases. Surveillance mechanisms acting at the molecular, organellar, and cellular level monitor mitochondrial integrity and ensure the maintenance of mitochondrial proteostasis. Here we will review the central role of mitochondrial chaperones and proteases, the cytosolic ubiquitin-proteasome system, and the mitochondrial unfolded response in this interconnected quality control network, highlighting the dual function of some proteases in protein quality control within the organelle and for the regulation of mitochondrial fusion and mitophagy.