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.
Mitochondrial complex I is the largest integral membrane enzyme of the respiratory chain and consists of 44 different subunits encoded in the mitochondrial and nuclear genome. Its biosynthesis is a highly complicated and multifaceted process involving at least 14 additional assembly factors. How these subunits assemble into a functional complex I and where the assembly factors come into play is largely unknown. Here, we applied a dynamic complexome profiling approach to elucidate the assembly of human mitochondrial complex I and its further incorporation into respiratory chain supercomplexes. We delineate the stepwise incorporation of all but one subunit into a series of distinct assembly intermediates and their association with known and putative assembly factors, which had not been implicated in this process before. The resulting detailed and comprehensive model of complex I assembly is fully consistent with recent structural data and the remarkable modular architecture of this multiprotein complex.
Virtually every mammalian cell contains mitochondria. These double-membrane organelles continuously change shape and position and contain the complete metabolic machinery for the oxidative conversion of pyruvate, fatty acids, and amino acids into ATP. Mitochondria are crucially involved in cellular Ca 2+ and redox homeostasis and apoptosis induction. Maintenance of mitochondrial function and integrity requires an inside-negative potential difference across the mitochondrial inner membrane. This potential is sustained by the electron-transport chain (ETC). NADH:ubiquinone oxidoreductase or complex I (CI), the first and largest protein complex of the ETC, couples the oxidation of NADH to the reduction of ubiquinone. During this process, electrons can escape from CI and react with ambient oxygen to produce superoxide and derived reactive oxygen species (ROS). Depending on the balance between their production and removal by antioxidant systems, ROS may function as signaling molecules or induce damage to a variety of biomolecules or both. The latter ultimately leads to a loss of mitochondrial and cellular function and integrity. In this review, we discuss (a) the role of CI in mitochondrial functioning; (b) the composition, structure, and biogenesis of CI; (c) regulation of CI function; (d) the role of CI in ROS generation; and (e) adaptive responses to CI deficiency.
Although originally identified as putative negative regulators of the cell cycle, recent studies have demonstrated that the PHB proteins act as a chaperone in the assembly of subunits of mitochondrial respiratory chain complexes. The two PHB proteins, Phblp and Phb2p, are located in the mitochondrial inner membrane where they form a large complex that represents a novel type of membrane-bound chaperone. On the basis of its native molecular weight, the PHB-complex should contain 12-14 copies of both Phblp and Phb2p. The PHB complex binds directly to newly synthesised mitochondrial translation products and stabilises them against degradation by membrane-bound metalloproteases belonging to the family of mitochondrial triple-A proteins. Sequence homology assigns Phb1p and Phb2p to a family of proteins which also contains stomatins, HflKC, flotillins and plant defence proteins. However, to date only the bacterial HflKC proteins have been shown to possess a direct functional homology with the PHB complex. Previously assigned actions of the PHB proteins, including roles in tumour suppression, cell cycle regulation, immunoglobulin M receptor binding and apoptosis seem unlikely in view of any hard evidence in their support. Nevertheless, because the proteins are probably indirectly involved in ageing and cancer, we assess their possible role in these processes. Finally, we suggest that the original name for these proteins, the prohibitins, should be amended to reflect their roles as proteins that hold badly formed subunits, thereby keeping the nomenclature already in use but altering its meaning to reflect their true function more accurately.
The assembly of cytochrome-c oxidase was studied in human cells cultured in the presence of inhibitors of mitochondrial or cytosolic protein synthesis. Mitochondrial fractions were resolved using twodimensional PAGE (blue native PAGE and tricine/SDS/PAGE) and subsequent western blots were developed with monoclonal antibodies against specific subunits of cytochrome-c oxidase. Proteins were also visualized using metabolic labeling followed by two-dimensional electrophoresis and fluorography. These techniques allowed identification of two assembly intermediates of cytochrome-c oxidase. Assembly of the 13 subunits of cytochrome-c oxidase starts with the association of subunit I with subunit IV. Then a larger subcomplex is formed, lacking only subunits VIa and either VIIa or VIIb.Keywords : cytochrome-c oxidase; assembly; respiratory chain ; cultured human cell.Cytochrome-c oxidase (COX) is the terminal enzyme of the in a stable intermediate complex made up solely of nuclear-encoded subunits [10,11]. respiratory chain located in the inner mitochondrial membrane. The enzyme complex catalyses the oxidation of cytochrome c Basic questions regarding the assembly of COX remain to be resolved. What is the order of assembly of the subunits? How by molecular oxygen; the energy released in this reaction is used to translocate protons across the inner mitochondrial membrane. is assembly regulated and what are the rate-limiting steps? Answers to these questions are not only important to understand The proton gradient resulting from the activity of the different respiratory-chain complexes is used to drive ATP synthesis. the function and regulation of the enzyme, but they will also provide information on causes of COX deficiency in patients. Mammalian COX contains 13 subunits, three of which (COX I, II and III) are encoded by the mitochondrial genome, while the Most investigations of the assembly pathway of COX employed metabolic labeling followed by immunoprecipitation [8Ϫ remaining ten subunits (COX IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc and VIII) are nuclear-encoded [1Ϫ5].10, 12]. This approach may give erroneous results because there are differences in epitope availability in partially assembled The recent resolution of bovine heart COX by X-ray analysis [6, 7] not only revealed the exact topology of its subunits, but complexes or because there is selective loss of subunits during immunoprecipitation. We have used two-dimensional electroalso assists studies aimed at clarifying the reaction mechanism of the enzyme. Moreover, the quarternary structure of the com-phoresis in combination with translational inhibitors and metabolic labeling to analyze COX assembly. Two assembly intermeplex puts certain structural constraints on models regarding the assembly pathway of COX. To resolve the assembly process of diates could be detected. the enzyme, however, additional experimental approaches are required. Previous studies of the assembly pathway of COX have shown that pools of unassembled subunits exist and that EXPERIMENTAL PR...
Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified member of the acyl-CoA dehydrogenase family. It closely resembles very long-chain acyl-CoA dehydrogenase (VLCAD), involved in mitochondrial beta oxidation of long-chain fatty acids. Contrary to its previously proposed involvement in fatty acid oxidation, we describe a role for ACAD9 in oxidative phosphorylation. ACAD9 binds complex I assembly factors NDUFAF1 and Ecsit and is specifically required for the assembly of complex I. Furthermore, ACAD9 mutations result in complex I deficiency and not in disturbed long-chain fatty acid oxidation. This strongly contrasts with its evolutionary ancestor VLCAD, which we show is not required for complex I assembly and clearly plays a role in fatty acid oxidation. Our results demonstrate that two closely related metabolic enzymes have diverged at the root of the vertebrate lineage to function in two separate mitochondrial metabolic pathways and have clinical implications for the diagnosis of complex I deficiency.
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