Les A. Grivell

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 mitochondrial PHB complex: roles in mitochondrial respiratory complex assembly, ageing and degenerative disease.

Abstract. 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, Phb1p 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 Phb1p 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.

A structure for the yeast prohibitin complex: Structure prediction and evidence from chemical crosslinking and mass spectrometry

The mitochondrial prohibitin complex consists of two subunits (PHB1 of 32 kD and PHB2 of 34 kD), assembled into a membrane‐associated supercomplex of approximately 1 MD. A chaperone‐like function in holding and assembling newly synthesized mitochondrial polypeptide chains has been proposed. To further elucidate the function of this complex, structural information is necessary. In this study we use chemical crosslinking, connecting lysine side chains, which are well scattered along the sequence. Crosslinked peptides from protease digested prohibitin complexes were identified with mass spectrometry. From these results, spatial restraints for possible protein conformation were obtained. Many interaction sites between PHB1 and PHB2 were found, whereas no homodimeric interactions were observed. Secondary and tertiary structural predictions were made using several algorithms and the models best fitting the spatial restraints were selected for further evaluation. From the structure predictions and the crosslink data we derived a structural building block of one PHB1 and one PHB2 subunit, strongly intertwined along most of their length. The size of the complex implies that approximately 14 of these building blocks are present. Each unit contains a putative transmembrane helix in PHB2. Taken together with the unit building block we postulate a circular palisade‐like arrangement of the building blocks projecting into the intermembrane space.

Mitochondrial assembly in yeast

The yeast Saccharomyces cerevisiae is likely to be the first organism for which a complete inventory of mitochondrial proteins and their functions can be drawn up. A survey of the 340 or so proteins currently known to be localised in yeast mitochondria reveals the considerable investment required to maintain the organelles own genetic system, which itself contributes seven key components of the electron transport chain. Translation and respiratory complex assembly are particularly expensive processes, together requiring around 150 of the proteins so far known. Recent developments in both areas are reviewed and approaches to the identification of novel mitochondrial proteins are discussed.

Subunit homology between Escherichia coli, mitochondrial and chloroplast ribosomes

Abstract Interchange experiments between ribosomal subunits from spinach chloroplasts, yeast mitochondria and Escherichia coli have been per formed to obtain more information on possible homologies existing between them. Homology between bacterial and chloroplast ribosomes is high, since hybrid ribosomes containing chloroplast and E. coli subunits are active in polyphenylalanine synthesis directed by poly (U). Mitochondrial ribosomal subunits, in contrast, do not form hybrid ribosomes with either chloroplast or E. coli subunits.

Inhibition by ethidium bromide of mitochondrial protein synthesis programmed by imported poly(U)

Summary Poly(U) stimulates incorporation of phenylalanine by mitochondria isolated from Xenopus laevis , but is without effect on mitochondria from yeast, Tetrahymena , rat or chick liver. Poly(U)-stimulated activity is resistant to cycloheximide and ribonuclease, but partially sensitive to chloramphenicol, consistent with the idea that import, followed by translation of the poly(U) on mitochondrial ribosomes is occurring. Ethidium bromide blocks the incorporation of phenylalanine, without affecting uptake of [ 3 H]poly(U) into ribonuclease-resistant form, indicating a direct effect of this agent on mitochondrial translation. This effect should be taken into account in the interpretation of experiments involving the use of ethidium as a probe for the existence of nuclear mRNAs within mitochondria in vivo .