Prohibitin function within mitochondria: essential roles for cell proliferation and cristae morphogenesis

Prohibitins comprise an evolutionary conserved and ubiquitously expressed family of membrane proteins. Various roles in different cellular compartments have been proposed for prohibitin proteins. Recent experiments, however, identify large assemblies of two homologous prohibitin subunits, PHB1 and PHB2, in the inner membrane of mitochondria as the physiologically active structure. Mitochondrial prohibitin complexes control cell proliferation, cristae morphogenesis and the functional integrity of mitochondria. The processing of the dynamin-like GTPase OPA1, a core component of the mitochondrial fusion machinery, has been defined as a key process affected by prohibitins. The molecular mechanism of prohibitin function, however, remained elusive. The ring-like assembly of prohibitins and their sequence similarity with lipid raft-associated SPFH-family members suggests a scaffolding function of prohibitins, which may lead to functional compartmentalization in the inner membrane of mitochondria.     

Prohibitins and Ras2 protein cooperate in the maintenance of mitochondrial function during yeast aging

The yeast Saccharomyces cerevisiae has a finite replicative life span. Yeasts possess two prohibitins, Phb1p and Phb2p, in similarity to mammalian cells. These proteins are located in the inner mitochondrial membrane, where they are involved in the processing of newly-synthesized membrane proteins. We demonstrate that the elimination of one or both of the prohibitin genes in yeast markedly diminished the replicative life span of cells that lack fully-functional mitochondria, while having no effect on cells with functioning mitochondria. This deleterious effect was suppressed by the deletion of the RAS2 gene. The expression of PHB1 and PHB2 declined gradually up to 5-fold during the life span. Cells in which PHB1 was deleted in conjunction with the absence of a mitochondrial genome displayed remarkable changes in mitochondrial morphology, distribution, and inheritance. This loss of mitochondrial integrity was not seen in cells devoid of PHB1 but possessing an intact mitochondrial genome. In a subset of the cells, the changes in mitochondrial integrity were associated with increased production of reactive oxygen species, which co-localized with the altered mitochondria. The mitochondrial deficits described above were all suppressed by deletion of RAS2. Our data, together with published information, are interpreted to provide a unified view of the role of the prohibitins in yeast aging. This model posits that the key initiating event is a decline in mitochondrial function, which leads to progressive oxidative damage that is exacerbated in the absence of the prohibitins. This aggravation of the initial damage is ameliorated by the suppression of the production of mitochondrial proteins in the absence of Ras2p signaling of mitochondrial biogenesis.     

Formation of membrane-bound ring complexes by prohibitins in mitochondria

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.     

AAA proteases with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent degradation of inner membrane proteins in mitochondria.

The mechanism of selective protein degradation of membrane proteins in mitochondria has been studied employing a model protein that is subject to rapid proteolysis within the inner membrane. Protein degradation was mediated by two different proteases: (i) the m-AAA protease, a protease complex consisting of multiple copies of the ATP-dependent metallopeptidases Yta1Op (Afg3p) and Yta12p (Rcalp); and (ii) by Ymelp (Ytallp) that also is embedded in the inner membrane. Ymelp, highly homologous to Yta1Op and Yta12p, forms a complex of approximately 850 kDa in the inner membrane and exerts ATP-dependent metallopeptidase activity. While the m-AAA protease exposes catalytic sites to the mitochondrial matrix, Ymelp is active in the intermembrane space. The Ymelp complex was therefore termed 'i-AAA protease'. Analysis of the proteolytic fragments indicated cleavage of the model polypeptide at the inner and outer membrane surface and within the membrane-spanning domain. Thus, two AAA proteases with their catalytic sites on opposite membrane surfaces constitute a novel proteolytic system for the degradation of membrane proteins in mitochondria.     

Membrane protein turnover by the m-AAA protease in mitochondria depends on the transmembrane domains of its subunits

AAA proteases are membrane-bound ATP-dependent proteases that are present in eubacteria, mitochondria and chloroplasts and that can degrade membrane proteins. Recent evidence suggests dislocation of membrane-embedded substrates for proteolysis to occur in a hydrophilic environment; however, next to nothing is known about the mechanism of this process. Here, we have analysed the role of the membrane-spanning domains of Yta10 and Yta12, which are conserved subunits of the hetero-oligomeric m-AAA protease in the mitochondria of Saccharomyces cerevisiae. We demonstrate that the m-AAA protease retains proteolytic activity after deletion of the transmembrane segments of either Yta10 or Yta12. Although the mutant m-AAA protease is still capable of processing cytochrome c peroxidase and degrading a peripheral membrane protein, proteolysis of integral membrane proteins is impaired. We therefore propose that transmembrane segments of m-AAA protease subunits have a direct role in the dislocation of membrane-embedded substrates.     

Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins

Prohibitins are ubiquitous, abundant and evolutionarily strongly conserved proteins that play a role in important cellular processes. Using blue native electrophoresis we have demonstrated that human prohibitin and Bap37 together form a large complex in the mitochondrial inner membrane. This complex is similar in size to the yeast complex formed by the homologues Phb1p and Phb2p. In yeast, levels of this complex are increased on co-overexpression of both Phb1p and Phb2p, suggesting that these two proteins are the only components of the complex. Pulse-chase experiments with mitochondria isolated from phb1/phb2-null and PHB1/2 overexpressing cells show that the Phb1/2 complex is able to stabilize newly synthesized mitochondrial translation products. This stabilization probably occurs through a direct interaction because association of mitochondrial translation products with the Phb1/2 complex could be demonstrated. The fact that Phb1/2 is a large multimeric complex, which provides protection of native peptides against proteolysis, suggests a functional homology with protein chaperones with respect to their ability to hold and prevent misfolding of newly synthesized proteins.     

The shortened replicative life span of prohibitin mutants of yeast appears to be due to defective mitochondrial segregation in old mother cells

Prohibitin proteins have been implicated in cell proliferation, aging, respiratory chain assembly and the maintenance of mitochondrial integrity. The prohibitins of Saccharomyces cerevisiae, Phb1 and Phb2, have strong sequence similarity with their human counterparts prohibitin and BAP37, making yeast a good model organism in which to study prohibitin function. Both yeast and mammalian prohibitins form high-molecular-weight complexes (Phb1/2 or prohibitin/BAP37, respectively) in the inner mitochondrial membrane. Expression of prohibitins declines with senescence, both in mammalian fibroblasts and in yeast. With a total loss of prohibitins, the replicative (budding) life span of yeast is reduced, whilst the chronological life span (the survival of stationary cells over time) is relatively unaffected. This effect of prohibitin loss on the replicative life span is still apparent in the absence of an assembled respiratory chain. It also does not reflect the production of extrachromosomal ribosomal DNA circles (ERCs), a genetic instability thought to be a major cause of replicative senescence in yeast. Examination of cells containing a mitochondrially targeted green fluorescent protein indicates this shortened life span is a reflection of defective mitochondrial segregation from the mother to the daughter in the old mother cells of phb mutant strains. Old mother phb mutant cells display highly aberrant mitochondrial morphology and, frequently, a delayed segregation of mitochondria to the daughter. They often arrest growth with their last bud strongly attached and with the mitochondria adjacent to the septum between the mother and the daughter cell.     

Prohibitin Family Members Interact Genetically with Mitochondrial Inheritance Components in Saccharomyces cerevisiae

Phb2p, a homolog of the tumor suppressor protein prohibitin, was identified in a genetic screen for suppressors of the loss of Mdm12p, a mitochondrial outer membrane protein required for normal mitochondrial morphology and inheritance in Saccharomyces cerevisiae. Phb2p and its homolog, prohibitin (Phb1p), were localized to the mitochondrial inner membrane and characterized as integral membrane proteins which depend on each other for their stability. In otherwise wild-type genetic backgrounds, null mutations in PHB1 and PHB2 did not confer any obvious phenotypes. However, loss of function of either PHB1 or PHB2 in cells with mitochondrial DNA deleted led to altered mitochondrial morphology, and phb1 or phb2 mutations were synthetically lethal when combined with a mutation in any of three mitochondrial inheritance components of the mitochondrial outer membrane, Mdm12p, Mdm10p, and Mmm1p. These results provide the first evidence of a role for prohibitin in mitochondrial inheritance and in the regulation of mitochondrial morphology.     

OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria

The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria.     

Prohibitins Are Required for Cancer Cell Proliferation and Adhesion

Prohibitin 1 (PHB1) is a highly conserved protein that together with its homologue prohibitin 2 (PHB2) mainly localizes to the inner mitochondrial membrane. Although it was originally identified by its ability to inhibit G1/S progression in human fibroblasts, its role as tumor suppressor is debated. To determine the function of prohibitins in maintaining cell homeostasis, we generated cancer cell lines expressing prohibitin-directed shRNAs. We show that prohibitin proteins are necessary for the proliferation of cancer cells. Down-regulation of prohibitin expression drastically reduced the rate of cell division. Furthermore, mitochondrial morphology was not affected, but loss of prohibitins did lead to the degradation of the fusion protein OPA1 and, in certain cancer cell lines, to a reduced capability to exhibit anchorage-independent growth. These cancer cells also exhibited reduced adhesion to the extracellular matrix. Taken together, these observations suggest prohibitins play a crucial role in adhesion processes in the cell and thereby sustaining cancer cell propagation and survival.     

Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and Leads to Tau Hyperphosphorylation and Neurodegeneration

Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegeneration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds. In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the dynamin-like GTPase OPA1 (optic atrophy 1), which mediates mitochondrial inner membrane fusion and cristae morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus, demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1, affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability, mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins. Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for tau pathologies caused by a dysfunction of mitochondria and raise the possibility that tau pathologies are associated with other neurodegenerative disorders caused by deficiencies in mitochondrial dynamics.     

Stimulation of mitogenic pathways through kinase-impaired mutants of the epidermal growth factor receptor

The two prohibitin proteins, Phb1p and Phb2p(BAP37), have been ascribed various functions, including cell cycle regulation, apoptosis, assembly of mitochondrial respiratory chain enzymes, and aging. We show that the mammalian prohibitins are present in the inner mitochondrial membrane and are always bound to each other, with no free protein detectable. They are coexpressed during development and in adult mammalian tissues, and expression levels are indicative of a role in mitochondrial metabolism, but are not compatible with roles in the regulation of cellular proliferation or apoptosis. High level expression of the proteins is consistently seen in primary human tumors, while cellular senescence of human and chick fibroblasts is accompanied by heterogeneous decreases in both proteins. The two proteins are induced by metabolic stress caused by an imbalance in the synthesis of mitochondrial- and nuclear-encoded mitochondrial proteins, but do not respond to oxidative stress, heat shock, or other cellular stresses. The gene promoter sequences contain binding sites for the Myc oncoprotein and overexpression of Myc induces expression of the prohibitins. The data support conserved roles for the prohibitins in regulating mitochondrial respiratory activity and in aging.     

The mitochondrial prohibitin complex is essential for embryonic viability and germline function in Caenorhabditis elegans

Prohibitins in eukaryotes consist of two subunits (PHB1 and PHB2) that together form a high molecular weight complex in the mitochondrial inner membrane. The evolutionary conservation and the ubiquitous expression in mammalian tissues of the prohibitin complex suggest an important function among eukaryotes. The PHB complex has been shown to play a role in the stabilization of newly synthesized subunits of mitochondrial respiratory enzymes in the yeast Saccharomyces cerevisiae. We have used Caenorhabditis elegans as model system to study the role of the PHB complex during development of a multicellular organism. We demonstrate that prohibitins in C. elegans form a high molecular weight complex in the mitochondrial inner membrane similar to that of yeast and humans. By using RNA-mediated gene inactivation, we show that PHB proteins are essential during embryonic development and are required for somatic and germline differentiation in the larval gonad. We further demonstrate that a deficiency in PHB proteins results in altered mitochondrial biogenesis in body wall muscle cells. This paper reports a strong loss of function phenotype for prohibitin gene inactivation in a multicellular organism and shows for the first time that prohibitins serve an essential role in mitochondrial function during organismal development.     

Oma1, a novel membrane-bound metallopeptidase in mitochondria with activities overlapping with the m-AAA protease

The integrity of the inner membrane of mitochondria is maintained by a membrane-embedded quality control system that ensures the removal of misfolded membrane proteins. Two ATP-dependent AAA proteases with catalytic sites at opposite membrane surfaces are key components of this proteolytic system. Here we describe the identification of a novel conserved metallopeptidase that exerts activities overlapping with the m-AAA protease and was therefore termed Oma1. Both peptidases are integral parts of the inner membrane and mediate the proteolytic breakdown of a misfolded derivative of the polytopic inner membrane protein Oxa1. The m-AAA protease cleaves off the matrix-exposed C-terminal domain of Oxa1 and processively degrades its transmembrane domain. In the absence of the m-AAA protease, proteolysis of Oxa1 is mediated in an ATP-independent manner by Oma1 and a yet unknown peptidase resulting in the accumulation of N- and C-terminal proteolytic fragments. Oma1 exposes its proteolytic center to the matrix side; however, mapping of Oma1 cleavage sites reveals clipping of Oxa1 in loop regions at both membrane surfaces. These results identify Oma1 as a novel component of the quality control system in the inner membrane of mitochondria. Proteins homologous to Oma1 are present in higher eukaryotic cells, eubacteria and archaebacteria, suggesting that Oma1 is the founding member of a conserved family of membrane-embedded metallopeptidases.     

The PHB1/2 phosphocomplex is required for mitochondrial homeostasis and survival of human T cells

Many immune pathologies are the result of aberrant regulation of T lymphocytes. A functional proteomics approach utilizing two-dimensional gel electrophoresis coupled with mass spectrometry was employed to identify differentially expressed proteins in response to T cell activation. Two members of the prohibitin family of proteins, Phb1 and Phb2, were determined to be up-regulated 4-5-fold upon activation of primary human T cells. Furthermore, their expression was dependent upon CD3 and CD28 signaling pathways that synergistically led to the up-regulation (13-15-fold) of Phb1 and Phb2 mRNA levels as early as 48 h after activation. Additionally, orthophosphate labeling coupled with phosphoamino acid analysis identified Phb1 to be serine and Phb2 serine and tyrosine phosphorylated. Tyrosine phosphorylation of Phb2 was mapped to Tyr248 using mass spectrometry and confirmed by mutagenesis and phosphospecific antibodies. In contrast to previous reports of Phb1 and Phb2 being nuclear localized, subcellular fractionation, immunofluorescent, and electron microscopy revealed both proteins to localize to the mitochondrial inner membrane of human T cells. Accordingly, small interfering RNA-mediated knockdown of Phbs in Kit225 cells resulted in disruption of mitochondrial membrane potential. Additionally, Phb1 and Phb2 protein levels were up-regulated 2.5-fold during cytokine deprivation-mediated apoptosis of Kit225 cells, suggesting this complex plays a protective role in human T cells. Taken together, Phb1 and Phb2 are novel phosphoproteins up-regulated during T cell activation that function to maintain mitochondrial integrity and thus represent previously unrecognized therapeutic targets for regulating T cell activation, differentiation, viability, and function.     

Yeast replicative life span--the mitochondrial connection

Mitochondria have been associated with aging in many experimental systems through the damaging action of reactive oxygen species. There is more, however, to the connection between mitochondria and Saccharomyces cerevisiae longevity and aging. Induction of the retrograde response, a pathway signaling mitochondrial dysfunction, results in the extension of life span and postponement of the manifestations of aging, changing the metabolic and stress resistance status of the cell. A paradox associated with the retrograde response is the simultaneous triggering of extrachromosomal ribosomal DNA circle (ERC) production, because of the deleterious effect these circles have on yeast longevity. The retrograde response gene RTG2 appears to play a pivotal role in ERC production, linking metabolism and genome stability. In addition to mother cell aging, mitochondria are important in establishment of age asymmetry between mother and daughter cells. The results more generally point to the existence of a mechanism to "filter" damaged components from daughter cells, a form of checkpoint control. Mitochondrial integrity is affected by the PHB1 and PHB2 genes, which encode inner mitochondrial membrane chaperones called prohibitins. The Phb1/2 proteins protect the cell from imbalances in the production of mitochondrial proteins. Such imbalances appear to cause a stochastic stratification of the yeast population with the appearance of short-lived cells. Ras2p impacts this process. Maintenance of mitochondrial membrane potential and the provision of Krebs cycle intermediates for biosyntheses appear to be crucial elements in yeast longevity. In sum, it is clear that mitochondria lie at the nexus of yeast longevity and aging.     

m-AAA protease-driven membrane dislocation allows intramembrane cleavage by rhomboid in mitochondria

Maturation of cytochrome c peroxidase (Ccp1) in mitochondria occurs by the subsequent action of two conserved proteases in the inner membrane: the m-AAA protease, an ATP-dependent protease degrading misfolded proteins and mediating protein processing, and the rhomboid protease Pcp1, an intramembrane cleaving peptidase. Neither the determinants preventing complete proteolysis of certain substrates by the m-AAA protease, nor the obligatory requirement of the m-AAA protease for rhomboid cleavage is currently understood. Here, we describe an intimate and unexpected functional interplay of both proteases. The m-AAA protease mediates the ATP-dependent membrane dislocation of Ccp1 independent of its proteolytic activity. It thereby ensures the correct positioning of Ccp1 within the membrane bilayer allowing intramembrane cleavage by rhomboid. Decreasing the hydrophobicity of the Ccp1 transmembrane segment facilitates its dislocation from the membrane and renders rhomboid cleavage m-AAA protease-independent. These findings reveal for the first time a non-proteolytic function of the m-AAA protease during mitochondrial biogenesis and rationalise the requirement of a preceding step for intramembrane cleavage by rhomboid.     

Translating m-AAA protease function in mitochondria to hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is a genetically heterogeneous neurodegenerative disorder that is characterized by progressive and cell-specific axonal degeneration. An autosomal recessive form of the disease is caused by mutations in paraplegin, which is a conserved subunit of the ubiquitous and ATP-dependent m-AAA protease in mitochondria. The m-AAA protease carries out protein quality control in the inner membrane of the mitochondria, suggesting a pathogenic role of misfolded proteins in HSP. A recent study demonstrates that the m-AAA protease regulates ribosome assembly and translation within mitochondria by controlling proteolytic maturation of a ribosomal subunit. Here, we will discuss implications of the dual role of the m-AAA protease in protein activation and degradation for mitochondrial dysfunction and axonal degeneration.