Journal of bioenergetics

Recent progress is summarized on the mechanism of phototransduction by sensory rhodopsin I (SR-I), a phototaxis receptor inHalobacterium halobium. Two aspects are emphasized: (i)The coupling of retinal isomerization to protein conformational changes. Retinal analogs have been used to probe chromophore-apoprotein interactions during the receptor activation process. One of the most important results is the finding of a steric trigger deriving from the interaction of residues on the protein with a methyl group near the isomerizing bond of the retinal (at carbon 13). Recent work on molecular genetic methods to further probe structure/function includes the synthesis and expression of an SR-I apoprotein gene designed for residue replacements by cassette mutagenesis, and transformation of anH. halobium mutant lacking all retinylidene proteins known in this species to SR-I+ and bacteriorhodopsin (BR)+. (ii)The relay of the SR-I signal to a post-receptor component. A carboxylmethylated protein (“MPP-I”) associated with SR-I and found in theH. halobium membrane exhibits homology with the signaling domain of eubacterial chemotaxis transducers (e.g.,Escherichia coli Tar, Tsr, and Trg proteins), suggesting a model based on SR-I → MPP-I signal relay.

Mitochondria undergo a permeability transition (PT), i.e., become nonselectively permeable to small solutes, in response to a wide range of conditions/compounds. In general, opening of the permeability transition pore (PTP) is Ca2+- and Pi-dependent and is blocked by cyclosporin A (CsA), trifluoperazine (TFP), ADP, and butylated hydroxytoluene (BHT). Gudz and coworkers have reported [7th European Bioenergetics Conference, EBEC Short Reports (1992)7, 125], however, that, under some conditions, BHT increases mitochondrial permeability via a process that may not share all of these characteristics. Specifically, they determined that the BHT-induced permeability transition was independent of Ca2+ and was insensitive to CsA. We have used mitochondrial swelling to compare in greater detail the changes in permeability induced by BHT and by Ca2+ plus Pi with the following results. (1) The dependence of permeability on BHT concentration is triphasic: there is a threshold BHT concentration (ca. 60 nmol BHT/ mg mitochondrial protein) below which no increase occurs; BHT enhances permeability in an intermediate concentration range; and at high BHT concentrations (> 120 nmol/mg) permeability is again reduced. (2) The effects of BHT depend on the ratio of BHT to mitochondrial protein. (3) Concentrations of BHT too low to induce swelling block the PT induced by Ca2+ and Pi. (4) The dependence of the Ca2+-triggered PT on Pi concentration is biphasic. Below a threshold of 50–100 ΜM, no swelling occurs. Above this threshold swelling increases rapidly. (5) Pi levels too low to support the Ca2+-induced PT inhibit BHT-induced swelling. (6) Swelling induced by BHT can bestimulated by agents and treatments that block the PT induced by Ca2+ plus Pi. These data suggest that BHT and Ca2+ plus Pi, increase mitochondrial permeability via two mutually exclusive mechanisms.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that causes motor neuron degeneration, progressive skeletal muscle atrophy, paralysis, and death. To understand the mechanism of motor neuron degeneration, we have analyzed the clinical disease progression and the pathological changes in a transgenic mouse model for ALS. We found massive mitochondrial vacuolation at the onset of disease. By detailed morphological observations, we have determined that this mitochondrial vacuolation is developed from expansion of mitochondrial intermembrane space and extension of the outer membrane and involves peroxisomes. Lysosomes do not actively participate at all stages of this vacuolation. We conclude that this mitochondrial vacuolation is neither classical mitochondrial permeability transition nor autophagic vacuolation. Thus, this appears to be a new form of mitochondrial vacuolation and we term this as mitochondrial vacuolation by intermembrane space expansion or MVISE.

Addition of Pb2+ to rat kidney mitochondria is followed by induction of several reactions: inhibition of Ca2+ uptake, collapse of the transmembrane potential, oxidation of pyridine nucleotides, and a fast release of accumulated Ca2+. When the incubation media are supplemented with ruthenium red, the effect of Pb2+ on NAD(P)H oxidation, membrane ΔΨ, and Ca2+ release are not prevented if malate-glutamate are the oxidizing substrates; however, the latter two lead-induced reactions are prevented by ruthenium red if succinate is the electron donor. It is proposed that in mitochondria oxidizing NAD-dependent substrates, Pb2+ induces Ca2+ release by promoting NAD(P)H oxidation and a parallel drop in ΔΨ due to its binding to thiol groups, located in the cytosol side of the inner membrane. In addition, it is proposed that with succinate as substrate, the Ca2+-releasing effect of lead is due to the collapse of the transmembrane potential as a consequence of the uptake of Pb2+ through the calcium uniporter, since such effect is ruthenium red sensitive.

During the last few years our knowledge of the structure and function of heme copper oxidases has greatly profited from the use of site-directed mutagenesis in combination with biophysical techniques. This, together with the recently-determined crystal structures of cytochrome c oxidase, has now made it possible to design experiments aimed at targeting specific pump mechanisms. Here, we summarize results from our recent kinetic studies of electron and proton-transfer reactions in wild-type and mutant forms of cytochrome c oxidase from Rhodobacter sphaeroides. These studies have made it possible to identify amino acid residues involved in proton transfer during specific reaction steps and provide a basis for discussion of mechanisms of electron and proton transfer in terminal oxidases. The results indicate that the pathway through K(I-362)/T(I-359), but not through D(I-132)/E(I-286), is used for proton transfer to a protonatable group interacting electrostatically with heme a 3, i.e., upon reduction of the binuclear center. The pathway through D(I-132)/E(I-286) is used for uptake of pumped and substrate protons during the pumping steps during O2 reduction.

In the secretion of polypeptides from Gram-negative bacteria, the outer membrane constitutes a specific barrier which has to be circumvented. In the majority of systems, secretion is two-step process, with initial export to the periplasm involving an N-terminal signal sequence. Transport across the outer membrane then involves a variable number of ancillary polypeptides including both periplasmic and outer membrane. While such ancillary proteins are probably specific for each secreted protein, the mechanism of movement across the outer membrane is unknown. In contrast to these systems, secretion of theE. coli hemolysin (HlyA) has several distinctive features. These include a novel targeting signal located within the last 50 or so C-terminal amino acids, the absence of any periplasmic intermediates in transfer, and a specific membrane-bound translocator, HlyB, with important mammalian homologues such as P-glycoprotein (Mdr) and the cystic fibrosis protein. In this review we discuss the nature of the HlyA targeting signal, the structure and function of HlyB, and the probability that HlyA is secreted directly to the medium through a trans-envelope complex composed of HlyB and HlyD.

The voltage dependent anion-channel, VDAC, is the major constitutive protein of the outer membrane of mitochondria. Functionally, VDAC is involved in the exchange of small metabolites over the mitochondrial outer membrane and supports enzymes of the cytoplasm with energy precursors i.e. ATP. Moreover, the channel alone or in complex with proteins of the inner mitochondrial membrane or the intermembrane space provides a basis for docking of cytosolic proteins which can regulate outer membrane permeability in several ways. Structurally, this channel has a bacterial origin by evolution and partly resembles bacterial porin functions. However, the structure seems more complex as a variety of interactions on both channel sides can occur. Therefore, our work described is aiming to determine the structure of VDAC at atomic resolution and together with functional data to understand better how this channel can carry out such a variety of differing functions.

In liver mitochondria isolated from hypothyroid rats, the rate of ATP synthesis is lower than in mitochondria from normal rats. Oligomycin-sensitive ATP hydrolase activity and passive proton permeability were significantly lower in submitochondrial particles from hypothyroid rats compared to those isolated from normal rats. In mitochondria from hypothyroid rats, the changes in catalytic activities of F0F1-ATP synthase are accompanied by a decrease in the amount of immunodetected β-F1, F01-PVP, and OSCP subunits of the complex. Northern blot hybridization shows a decrease in the relative cytosolic content of mRNA for β-F1 subunit in liver of hypothyroid rats. Administration of 3,5,3′-triodo-L-thyronine to the hypothyroid rats tends to remedy the functional and structural defects of F0F1-ATP synthase observed in the hypothyroid rats. The results obtained indicate that hypothyroidism leads to a decreased expression of F0F1-ATP synthase complex in liver mitochondria and this contributes to the decrease of the efficiency of oxidative phosphorylation.