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

Abstract

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.

Fig. 1. Prohibitin and BAP 37 form a high molecular weight complex in human mitochondria. (A) Two-dimensional electrophoresis (BN and SDS–PAGE) of mitochondria extracted from human fibroblasts. The complexes were transferred to nitrocellulose and blots were incubated with antisera directed against prohibitin, BAP37 and human cytochrome c oxidase holo-enzyme (COX). Arrows indicate the directions of the first and second dimension. (B) A western blot of a first dimension BN electrophoresis gel of mitochondria of B2.ρ⁰ (ρ⁰) and wild-type A549 (wt) lung carcinoma cells. The blots were probed with polyclonal antibodies against prohibitin, BAP37 and COX.

Fig. 2. Phb1p and Phb2p form a 1 MDa complex of which they are the only components. (A) 2D PAGE of mitochondrial membranes of a W303 Δphb2 strain transformed with the multicopy plasmid YEplac195-PHB2-T7-tag. After western blotting, the blots were incubated with a monoclonal antibody recognizing the T7-tag and subsequently with the polyclonal antibody against Phb1p. (B) A Phb1p polyclonal antibody was used to check the expression level of the Phb1/2 complex. The Phb1 signal was only increased when both PHB1 and PHB2 were overexpressed (W303+mcP1+P2). COX is indicated as a reference protein, which could be demonstrated by incubating with the Cox1p monoclonal antibody. (C) When overexpressed [see (B)], the Phb1/Phb2 complex could also be detected after Coomassie staining. For better comparison mitochondrial ATP synthase (ATPase) is indicated.

Fig. 3. The Phb1/2 complex stabilizes mitochondrial translation products. Proteins were pulse-labelled with [³⁵S]methionine in isolated mitochondria for 10 and 30 min. After addition of a large excess of unlabelled methionine, mitochondria were chased for 30 and 60 min, respectively and analysed on SDS–PAGE (see Materials and methods). Mitochondrially translated products were compared in a disruptant strain (Δphb1/Δphb2) and in a strain overexpressing the Phb1/2 complex (Δphb1/Δphb2 mcP1+P2). The mitochondrial translation products Var1p, Cox1p, Cobp and Cox2p are indicated. Cox3p and Atp6p were not separated using these electrophoresis conditions.

Fig. 4. COX subunits associate temporarily with the Phb1/2 complex. (A) A 2D gel of mitochondrial membranes of cells overexpressing the Phb1/2 complex (W303+mcP1+P2) and cells lacking this complex (Δphb1/Δphb2), which were blotted to nitrocellulose. Western blots were first incubated with the monoclonal antibody against Cox3p, and subsequently with the polyclonal antibody against Phb1p. (B) The same strains were used in a pulse–chase experiment (see Materials and methods). After 40 min pulse labelling, cells were chased for 60 min. Mitochondrial pellets were run on 2D PAGE as described before. Labelled proteins were detected using a PhosphorImager. Cox3p is indicated.

Fig. 5. Cox2p and Cox3p specifically co-immunoprecipitate with the Phb1/Phb2 complex. Mitochondria from W303+mcP1+P2 (lanes 1 and 3) and Δphb1/Δphb2 (lanes 2 and 4) were used for immunoprecipitation with the polyclonal antibody against Phb1p. Lanes 1 and 2 represent 1% of the total lysate and lanes 3 and 4 correspond to the immunoprecipitate. The same blot was first incubated with the monoclonal antibodies directed against Cox3p and Cox2p (A) and subsequently with the polyclonal antibody against Phb1p (B). To demonstrate the specificity of the co-immunoprecipitate, a separate blot of an SDS gel containing total lysate (1) and the immunoprecipitate (3) was incubated with a polyclonal antibody against the mitochondrial ADP/ATP carrier protein (AAC) [(C), left panel] and additionally with the Phb1p antibody [(C), right panel].

Fig. 6. Sequence conservation between prohibitins and part of E.coli GroEL. Representative members of the prohibitin family were aligned using ClustalW (version 1.4) using values of 10 and 0.05 as penalties for gap opening and extension, respectively. The aligned sequences were then compared with the region of the sequence identified by Psi-Blast in the CPN60 chaperone family as displaying similarity (E-value of 5e^–50 after four iterations against the NCBI non-redundant protein database). Amino acids identical or chemically conserved between E.coli GroEL, as representative of the CPN60 family, and other sequences are highlighted by black and grey shading, respectively. Groups of chemically conserved amino acids are: (V,I,L,M); (F,W,Y); (E,D); (Q,N); (S,T); (R,K); (A,G); H; P; C. Database accession numbers and the positions of the sequences shown are: S.cerevisiae Phb1 SwissProt P40961, residues 73–287; human prohibitin SwissProt P35232, residues 71–272; Drosophila melanogaster prohibitin homologue SwissProt P24156, residues 13–203; S.cerevisiae Phb2 SwissProt P50085, residues 102–315; human B-cell-associated protein (BAP37) DDBJ/EMBL/GenBank AAF17231, residues 85–299; Arabidopsis thaliana prohibitin homologue, DDBJ/EMBL/GenBank AAC49691.1, residues 76–277; E.coli GroEL SwissProt P06139, residues 39–203.

Fig. 7. Phb1/Phb2 complex levels are increased in respiratory chain deficient mutants. Cells lacking the MSS51 gene (Δmss51) were compared with wild-type cells (W303). Mitochondrial membrane extracts were analysed by 2D (BN/SDS)–PAGE and the Phb1/Phb2 complex was detected with a polyclonal antibody against Phb1p. For better comparison, mitochondrial ATP synthase (ATPase) is indicated.