The respiratory chain of the thermophilic archaeon Sulfolobus metallicus: studies on the type-II NADH dehydrogenase

The membranes of the thermoacidophilic archaeon Sulfolobus metallicus exhibit an oxygen consumption activity of 0.5 nmol O(2) min(-1) mg(-1), which is insensitive to rotenone, suggesting the presence of a type-II NADH dehydrogenase. Following this observation, the enzyme was purified from solubilised membranes and characterised. The pure protein is a monomer with an apparent molecular mass of 49 kDa, having a high N-terminal amino acid sequence similarity towards other prokaryotic enzymes of the same type. It contains a covalently attached flavin, which was identified as being FMN by 31P-NMR spectroscopy, a novelty among type-II NADH dehydrogenases. Metal analysis showed the absence of iron, indicating that no FeS clusters are present in the protein. The average reduction potential of the FMN group was determined to be +160 mV, at 25 degrees C and pH 6.5, by redox titrations monitored by visible spectroscopy. Catalytically, the enzyme is a NADH:quinone oxidoreductase, as it is capable of transferring electrons from NADH to several quinones, including ubiquinone-1, ubiquinone-2 and caldariella quinone. Maximal turnover rates of 195 micromol NADH oxidized min(-1) mg(-1) at 60 degrees C were obtained using ubiquinone-2 as electron acceptor, after enzyme dilution and incubation with phospholipids.     

Collective chain dynamics in lipid bilayers by inelastic x-ray scattering

We investigated the application of inelastic x-ray scattering (IXS) to lipid bilayers. This technique directly measures the dynamic structure factor S(q,omega) which is the space-time Fourier transform of the electron density correlation function of the measured system. For a multiatomic system, the analysis of S(q,omega) is usually complicated. But for multiple bilayers of lipid, S(q,omega) is dominated by chain-chain correlations within individual bilayers. Thus IXS provides a unique probe for the collective dynamics of lipid chains in a bilayer that cannot be obtained by any other method. IXS of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylcholine + cholesterol at two different concentrations were measured. S(q,omega) was analyzed by three-mode hydrodynamic equations, including a thermal diffusive mode and two propagating acoustic modes. We obtained the dispersion curves for the phonons that represent the collective in-plane excitations of lipid chains. The effect of cholesterol on chain dynamics was detected. Our analysis shows the importance of having a high instrument resolution as well as the requirement of sufficient signal-to-noise ratio to obtain meaningful results from such an IXS experiment. The requirement on signal-to-noise also applies to molecular dynamics simulations.     

Misfolded plant virus proteins: elicitors and targets of ubiquitylation

Mutant tobacco mosaic virus (TMV) coat proteins (CPs) with known amino acid replacements provide well defined examples of destabilized tertiary structures. Here we show that misfolded TMV CPs, but not functional wild-type CPs, induce massive ubiquitylation in tobacco cells and that denatured, insoluble CP subunits are the main substrates of ubiquitin conjugation. As TMV CPs can be easily manipulated they are unique tools to study the molecular basis of the plant cell's response to aberrant protein structures and the associated intracellular stress reactions.     

Cis- and trans-acting influences on telomeric position effect in Drosophila melanogaster detected with a subterminal transgene

One model of telomeric position effect (TPE) in Drosophila melanogaster proposes that reporter genes in the vicinity of telomeres are repressed by subterminal telomere-associated sequences (TAS) and that variegation of these genes is the result of competition between the repressive effects of TAS and the stimulating effects of promoters in the terminal HeT-A transposon array. The data presented here support this model, but also suggest that TPE is more complex. Activity of a telomeric white reporter gene increases in response to deletion of some or all of the TAS on the homolog. Only transgenes next to fairly long HeT-A arrays respond to this trans-interaction. HeT-A arrays of 6-18 kb respond by increasing the number of dark spots on the eye, while longer arrays increase the background eye color or increase the number of spots sufficiently to cause them to merge. Thus, expression of a subtelomeric reporter gene is influenced by the telomere structure in cis and trans. We propose that the forces involved in telomere length regulation in Drosophila are the underlying forces that manifest themselves as TPE. In the wild-type telomere TAS may play an important role in controlling telomere elongation by repressing HeT-A promoter activity. Modulation of this repression by the homolog may thus regulate telomere elongation.     

Hrs and endocytic sorting of ubiquitinated membrane proteins

Endocytosed receptors are either recycled to the plasma membrane or trapped within intralumenal vesicles of multi-vesicular bodies for subsequent degradation in lysosomes. How the cell is able to sort receptors in endosomes has so far been largely unknown. The hepatocyte growth factor regulated tyrosine kinase substrate, Hrs, is an essential protein that has been implicated in cell signalling and intracellular membrane trafficking. Very recently, several reports have demonstrated a role for Hrs in endocytic sorting of ubiquitinated membrane proteins. Here, we review current knowledge about how Hrs recognises ubiquitinated cargo that is destined for lysosomal degradation, and how Hrs may act as a key regulator of the molecular machinery involved in receptor sorting and multivesicular body formation.     

Nonelemental visual learning in honeybees

Free-flying honeybees, Apis mellifera, learn visual stimuli in the appetitive context of food search. Visual compound stimuli are relevant in nautre as bees learn flower images that consist of many visual elements. We studied whether elemental associations between each visual element and the reinforcement (elemental approach) are enough to explain the solving of visual discrimination problems that raise ambiguity at the elemental level. We asked whether bees could solve three different visual discriminations: (1) positive patterning (A−, B−, AB+); (2) negative patterning (A+, B+, AB−); and (3) biconditional discrimination (AB+, CD+, AC−, BD−). In experiments 1 and 2 bees had to discriminate a yellow-violet chequerboard from the yellow or the violet squares alone. In experiment 3, four different gratings combining one colour (yellow or violet) with one orientation (vertical or horizontal) had to be discriminated. In all three problems binary compounds were trained in such a way that each element appeared equally often as rewarded and nonrewarded. Bees could solve the three discrimination problems. They always chose the reinforced stimulus despite ambiguity at the level of the elements. For solving positive patterning, elemental processing could be used. For negative patterning and biconditional discrimination, nonelemental processing strategies (unique-cue or configural approach) are necessary to account for these results. Although we cannot decide between a configural and a unique-cue interpretation, we can clearly reject purely elemental processing in these cases. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Presenilin-dependent intramembrane proteolysis of CD44 leads to the liberation of its intracellular domain and the secretion of an Abeta-like peptide

Alzheimer's disease (AD)-associated gamma-secretase is a presenilin (PS)- dependent proteolytic activity involved in the intramembraneous cleavage of the beta-amyloid precursor protein, Notch, LDL receptor-related protein, E-cadherin, and ErbB-4. This cut produces the corresponding intracellular domains (ICD), which are required for nuclear signaling of Notch and probably ErbB-4, the beta-amyloid precursor protein, E-cadherin, and the LDL receptor-related protein as well. We have now investigated CD44, a cell surface adhesion molecule, which also undergoes an intramembraneous cleavage to liberate its ICD. We demonstrate that this cleavage requires a PS-dependent gamma-secretase activity. A loss-of-function PS1 mutation, a PS1/PS2 knockout, as well as two independent and highly specific gamma-secretase inhibitors, abolish this cleavage. Surprisingly, small peptides similar to the amyloid beta-peptide (Abeta) are generated by an additional cut in the middle of the transmembrane region of CD44. Like Abeta, these CD44 beta-peptides are generated in a PS-dependent manner. These findings therefore suggest a dual intramembraneous cleavage mechanism mediated by PS proteins. The dual cleavage mechanism is required for nuclear signaling as well as removal of remaining transmembrane domains, a general function of PS in membrane protein metabolism.     

The binding of Xanthophylls to the bulk light-harvesting complex of photosystem II of higher plants. A specific requirement for carotenoids with a 3-hydroxy-beta-end group

The pigment composition of the light-harvesting complexes (LHCs) of higher plants is highly conserved. The bulk complex (LHCIIb) binds three xanthophyll molecules in combination with chlorophyll (Chl) a and b. The structural requirements for binding xanthophylls to LHCIIb have been examined using an in vitro reconstitution procedure. Reassembly of the monomeric recombinant LHCIIb was performed using a wide range of native and nonnative xanthophylls, and a specific requirement for the presence of a hydroxy group at C-3 on a single beta-end group was identified. The presence of additional substituents (e.g. at C-4) did not interfere with xanthophyll binding, but they could not, on their own, support reassembly. cis isomers of zeaxanthin, violaxanthin, and lutein were not bound, whereas all-trans-neoxanthin and different chiral forms of lutein and zeaxanthin were incorporated into the complex. The C-3 and C-3' diols lactucaxanthin (a carotenoid native to many plant LHCs) and eschscholtzxanthin (a retro-carotenoid) both behaved very differently from lutein and zeaxanthin in that they would not support complex reassembly when used alone. Lactucaxanthin could, however, be bound when lutein was also present, and it showed a high affinity for xanthophyll binding site N1. In the presence of lutein, lactucaxanthin was readily bound to at least one lutein-binding site, suggesting that the ability to bind to the complex and initiate protein folding may be dependent on different structural features of the carotenoid molecule. The importance of carotenoid end group structure and ring-to-chain conformation around the C-6-C-7 torsion angle of the carotenoid molecule in binding and complex reassembly is discussed.     

Observing structure,function and assembly of single proteins by AFM

Single molecule experiments provide insight into the individuality of biological macromolecules, their unique function, reaction pathways, trajectories and molecular interactions. The exceptional signal-to-noise ratio of the atomic force microscope allows individual proteins to be imaged under physiologically relevant conditions at a lateral resolution of 0.5–1 nm and a vertical resolution of 0.1–0.2 nm. Recently, it has become possible to observe single molecule events using this technique. This capability is reviewed on various water-soluble and membrane proteins. Examples of the observation of function, variability, and assembly of single proteins are discussed. Statistical analysis is important to extend conclusions derived from single molecule experiments to protein species. Such approaches allow the classification of protein conformations and movements. Recent developments of probe microscopy techniques allow simultaneous measurement of multiple signals on individual macromolecules, and greatly extend the range of experiments possible for probing biological systems at the molecular level. Biologists exploring molecular mechanisms will benefit from a burgeoning of scanning probe microscopes and of their future combination with molecular biological experiments.