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.

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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 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.Keywords: Crosslinking; mass spectrometry; prohibitin complex; PHB complex; structure predictionThe two structurally related proteins PHB1 and PHB2, previously referred to as prohibitin proteins, localize to mitochondria in mammals, plants, and yeast (Ikonen et al. 1995;Coates et al. 1997;Snedden and Fromm 1997;Berger and Yaffe 1998;Steglich et al. 1999;Nijtmans et al. 2002). In yeast, levels of PHB1 and PHB2 have shown to be interdependent, and have been shown to physically associate with each other to form a large multimeric complex in the mitochondrial inner membrane of mammals and yeast (Snedden and Fromm 1997;Steglich et al. 1999;Nijtmans et al. 2000). The molecular mass of the so-called PHB complex is estimated to be 1 MD by migration in Blue Native Electrophoresis (BNE) experiments. PHB constituent proteins are ubiquitously expressed in mammalian tissues, and have been highly conserved through evolution, suggesting a vital function among eukaryotes. To date, various functions have been attributed to both PHB proteins, including cell cycle Reprint requests to: Jaap Willem Back, SILS/Mass Spectrometry Group, Nieuwe Achtergracht 166, 1018WV Amsterdam, The Netherlands; e-mail: jwback@science.uva.nl; fax: 31(20)5256568.Abbreviations: PHB, prohibitin; DTSP, dithiobis(succinimidylpropionate); sBID,sulfo-N-benzyliminodiacetoylhydroxysuccinimid; BNE, blue native gel electrophoresis; MALDI, matrix-assisted laser desorption ionization; ESI, electrospray ionization; TOF, time of flight; MS, mass spectrometry; MSMS, low energy collision experiments; CID, collision-induced dissociatio...

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