Anton O. Muijsers

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.

Chemical cross-linking and mass spectrometry for protein structural modeling

The growth of gene and protein sequence information is currently so rapid that three-dimensional structural information is lacking for the overwhelming majority of known proteins. In this review, efforts towards rapid and sensitive methods for protein structural characterization are described, complementing existing technologies. Based on chemical cross-linking and offering the analytical speed and sensitivity of mass spectrometry these methodologies are thought to contribute valuable tools towards future high throughput protein structure elucidation.

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.

hnRNP proteins and B23 are the major proteins of the internal nuclear matrix of HeLa S3 cells

The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina‐pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa S3 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high‐resolution two‐dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA.

The soluble NAD+-Reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH.

The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H(2). We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).

Biochemical and biophysical studies on cytochrome aa3. V. Binding of cyanide to cytochrome aa3

Abstract 1. 1. The effect of cyanide on the enzymic activity of cytochrome aa 3 shows that 1 mole cyanide is tightly bound to 1 mole cytochrome aa 3 . This is confirmed by the isolation of this complex (cyano-cytochrome aa 3 ). 2. 2. The rate constant for cyanide binding is 2 M −1 · sec −1 (pH 8.0, 0). From the K D of 7 · 10 −7 M (obtained from equilibrium dialyses), a dissociation rate constant of 1.4 · 10 −6 sec −1 is calculated. 3. 3. The time needed for equilibration of cyanide and cytochrome aa 3 depends on the redox state of the enzyme. 4. 4. Under conditions of reducing preincubation with ascorbate and cytochrome c the inhibition is noncompetitive towards cytochrome c with a K i of 8 · 10 −8 –9 × 10 −8 M. 5. 5. In the presence of reducing equivalents cyanide dissociates readily from cyano-cytochrome aa 3 to form enzymically active cytochrome aa 3 . The dissociation rate constant is 2 · 10 −3 sec −1 (pH 6.0, 25°). 6. 6. It is suggested, that the cavity in which the haem of cytochrome aa 3 is buried is more closed in the oxidized than in the reduced form and that the conformation is determined by the redox state of cytochrome a .

A new crosslinker for mass spectrometric analysis of the quaternary structure of protein complexes.

Mass spectrometric structural analysis of crosslinked peptides is a powerful method to elucidate the spatial arrangement of polypeptides in protein complexes. Our aim is to develop bifunctional crosslinkers that, after crosslinking protein complexes followed by proteolytic digestion, give rise to crosslinked peptides that can be readily tracked down by mass spectrometry. To this end we synthesized the crosslinker N-benzyliminodiacetoyloxysuccinimid (BID), which yields stable benzyl cation marker ions upon low-energy collisioninduced dissociation (CID) tandem mass spectrometry. Sensitive detection of the marker ion upon low-energy CID is demonstrated with different BID-crosslinked peptide preparations. With BID it becomes possible to retrieve crosslinked and crosslinker-adducted peptides, without the necessity of purifying crosslinked peptides prior to identification. The basic design of this crosslinker can be varied upon, in order to meet specific crosslinking needs.

Biochemical and biophysical studies on cytochrome c oxidase. X. Spectral and potentiometric properties of the hemes and coppers

Abstract 1. 1. Potentiometric titrations of highly purified cytochrome c oxidase, carried out in the absence of cytochrome c at the wavelengths 410, 424, 445 and 605 nm, show that both heme a groups act as independent one-electron acceptors with the same midpoint potential ( E 0 ′ = 280 mV, n = 1.0). The titration of the copper atoms at 830 nm suggests an equilibrium between one- and two-electron acceptors ( E 0 ′ = 280 mV, n = 1.6). 2. 2. When cytochrome c is present, the copper atoms titrate as single-electron acceptors ( E 0 ′ = 280 mV, n = 1.0). Two heme a groups with different midpoint potentials ( E 0 ′ = 370 mV, n = 1.0 and E 0 ′ = 230 mV, n = 1.0) contribute about equally to the absorbance differences on reduction at 410, 424, 445 and 605 nm. 3. 3. Both in the absence and presence of cytochrome c the difference spectrum (reduced minus oxidized) of the heme a group that is reduced at high oxidation-reduction potentials peaks at 603.5 and 444 nm, whilst that of the heme a group that is reduced at low redox potentials peaks at 607 and 446 nm. This indicates that the difference spectrum of one of the heme a groups depends on the redox state of the other. 4. 4. No evidence is found for Lembergs ( Physiol. Rev. 49 (1969) 48–121) suggestion that cytochrome a 3 absorbs maximally at 411 nm and cytochrome a at 423 nm. 5. 5. It is concluded that the heme a groups in cytochrome c oxidase, in the absence of inhibitors such as CO, azide and cyanide, are formally equivalent.

R174 of Escherichia coli FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division

We investigated the interaction between FtsZ and the cytoplasmic membrane using inside‐out vesicles. Comparison of the trypsin accessibility of purified FtsZ and cytoplasmic membrane‐bound FtsZ revealed that the protruding loop between helix 6 and helix 7 is protected from trypsin digestion in the latter. This hydrophobic loop contains an arginine residue at position 174. To investigate the role of R174, this residue was replaced by an aspartic acid, and FtsZ‐R174D was fused to green fluorescent protein (GFP). FtsZ‐R174D‐GFP could localize in an FtsZ and in an FtsZ84(Ts) background at both the permissive and the non‐permissive temperature, and it had a reduced affinity for the cytoplasmic membrane compared with wild‐type FtsZ. FtsZ‐R174D could also localize in an FtsZ depletion strain. However, in contrast to wild‐type FtsZ, FtsZ‐R174D was not able to complement the ftsZ84 mutation or the depletion strain and induced filamentation. In vitro polymerization experiments showed that FtsZ‐R174D is able to polymerize, but that these polymers cannot form bundles in the presence of 10 mM CaCl2. This is the first description of an FtsZ mutant that has reduced affinity for the cytoplasmic membrane and does not support cell division, but is still able to localize. The mutant is able to form protofilaments in vitro but fails to bundle. It suggests that neither membrane interaction nor bundling is a requirement for initiation of cell division.

CHARACTERIZATION OF THE RESPIRATORY CHAIN FROM CULTURED CRITHIDIA FASCICULATA

Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplastids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia fasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of F0F1-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial F0F1-ATPase. Amino acid sequence analysis, combined with binding studies using dicyclohexyldiimide and azido ATP allowed the identification of two F0 subunits (b and c) and all of the F1 subunits. The F0 b subunit has a low degree of similarity to subunit b from other organisms. The F1 alpha subunit is extremely small making the beta subunit the largest F1 subunit. Other respiratory-chain complexes were also analyzed. Interestingly, an NADH: ubiquinone oxidoreductase (complex I) appeared to be absent, as judged by electron paramagnetic resonance (EPR), enzyme activity and 2D PAGE analysis. Cytochrome c oxidase (complex IV) displayed a subunit pattern identical to that reported for the purified enzyme, whereas cytochrome c reductase (complex III) appeared to contain two extra subunits. A putative complex II was also identified. The amino acid sequences of the subunits of these complexes also show a very low degree of similarity (if any) to the corresponding sequences in other organisms. Remarkably, peptide sequences derived from mitochondrially encoded subunits were not found in spite of the fact that sequences were obtained of virtually all subunits of complex III, IV and V.