Mitochondrial import driving forces: enhanced trapping by matrix Hsp70 stimulates translocation and reduces the membrane potential dependence of loosely folded preproteins

The mitochondrial heat shock protein Hsp70 (mtHsp70) is essential for driving translocation of preproteins into the matrix. Two models, trapping and pulling by mtHsp70, are discussed, but positive evidence for either model has not been found so far. We have analyzed a mutant mtHsp70, Ssc1-2, that shows a reduced interaction with the membrane anchor Tim44, but an enhanced trapping of preproteins. Unexpectedly, at a low inner membrane potential, ssc1-2 mitochondria imported loosely folded preproteins more efficiently than wild-type mitochondria. The import of a tightly folded preprotein, however, was not increased in ssc1-2 mitochondria. Thus, enhanced trapping by mtHsp70 stimulates the import of loosely folded preproteins and reduces the dependence on the import-driving activity of the membrane potential, directly demonstrating that trapping is one of the molecular mechanisms of mtHsp70 action.     

The protein import motor of mitochondria: unfolding and trapping of preproteins are distinct and separable functions of matrix Hsp70.

Mitochondrial heat shock protein 70 (mtHsp70) functions in unfolding, translocation, and folding of imported proteins. Controversial models of mtHsp70 action have been discussed: (1) physical trapping of preproteins is sufficient to explain the various mtHsp70 functions, and (2) unfolding of preproteins requires an active motor function of mtHsp70 ("pulling"). Intragenic suppressors of a mutant mtHsp70 separate two functions: a nonlethal folding defect caused by enhanced trapping of preproteins, and a conditionally lethal unfolding defect caused by an impaired interaction of mtHsp70 with the membrane anchor Tim44. Even enhanced trapping in wild-type mitochondria does not generate a pulling force. The motor function of mtHsp70 cannot be explained by passive trapping alone but includes an essential ATP-dependent interaction with Tim44 to generate a pulling force and unfold preproteins.     

Uptake of antineoplastic agents into large unilamellar vesicles in response to a membrane potential

Many drugs exhibit lipophilic and cationic (basic) characteristics. Previous studies have shown that lipophilic cations can be accumulated into model membrane 'liposomal' (vesicular) systems in response to establishing a membrane potential (inside negative) across the vesicle membrane. We demonstrate here that the anticancer drugs, adriamycin and vinblastine, can be rapidly accumulated into egg phosphatidylcholine large unilamellar vesicles in response to a valinomycin-dependent K+ diffusion potential (delta psi) to achieve high effective interior concentrations. Further, trapping efficiencies approaching 100% can be easily achieved. The influence of lipid composition and the requirement for valinomycin have been examined for adriamycin. Equimolar cholesterol levels inhibit the uptake process at 20 degrees C. However, incubation at higher temperature results in enhanced uptake. Similarly, the presence of egg phosphatidylserine or incubation at elevated temperatures results in significant adriamycin uptake in the absence of valinomycin. It is shown that the adriamycin retention time in the vesicles is enhanced by an order of magnitude or more when actively trapped by the presence of a membrane potential in comparison to passive trapping procedures. It is suggested that such active trapping procedures may be of use for loading liposomal systems for drug delivery applications, and may provide avenues for controlled release of encapsulated material.     

A Method for the Removal from Membrane Filters of Micro-Organisms Filtered from Water and Air

S ummary : The micro-organisms in tap water were quantitatively (99%) retained by Oxoid membrane filters. The trapped organisms were extracted from the membrane filter by a simple washing process using glass beads or a magnetic stirrer. For counts on air, the membrane filter was considerably more efficient than an ammonium alginate filter in trapping bacteria, but both techniques were equally satisfactory for moulds.     

An integrated microfluidic device for single cell trapping and osmotic behavior investigation of mouse oocytes

Transport properties of oocytes play an important role in the optimization of their cryopreservation. However, there are still no systematical investigations on oocyte transport properties from the viewpoint of single-cell trapping and high precision perfusion, especially with the powerful microfluidic approach. To this end, we developed an easy-to-fabricate and easy-to-use microfluidic chip along with automatic single cell trapping capability to investigate the oocyte membrane transport properties. The experimental results indicate that the device is available and reliable. We further performed a comparative study of the oocyte membrane transport properties between single and multi-step CPA addition protocols and confirmed that the transport property parameters measured by single-step osmotic shift could not be used for prediction of the osmotic responses of oocytes in multi-step CPA addition. This study provides a powerful tool for investigation of oocyte osmotic responses.     

Binding of metallic ions to the outer membrane of Escherichia coli.

The binding of metal ions by the outer membrane of Escherichia coli K-12 strain AB264 was investigated by using outer membrane obtained after Triton X-100 extraction of purified cell envelopes. Binding studies, conducted under saturating conditions, indicated a selective trapping of certain metallic ions. Low-dose electron microscopy of metal-loaded samples revealed an aggregative deposition of lead on one surface of the membrane which suggests that at least one distinctive binding site is asymmetrically arranged in these outer membrane vesicles.     

Binding of metallic ions to the outer membrane of Escherichia coli

The binding of metal ions by the outer membrane of Escherichia coli K-12 strain AB264 was investigated by using outer membrane obtained after Triton X-100 extraction of purified cell envelopes. Binding studies, conducted under saturating conditions, indicated a selective trapping of certain metallic ions. Low-dose electron microscopy of metal-loaded samples revealed an aggregative deposition of lead on one surface of the membrane which suggests that at least one distinctive binding site is asymmetrically arranged in these outer membrane vesicles.     

Single-molecule manipulation of macromolecules on GUV or SUV membranes using optical tweezers

Despite their wide applications in soluble macromolecules, optical tweezers have rarely been used to characterize the dynamics of membrane proteins, mainly due to the lack of model membranes compatible with optical trapping. Here, we examined optical trapping and mechanical properties of two potential model membranes, giant and small unilamellar vesicles (GUVs and SUVs, respectively) for studies of membrane protein dynamics. We found that optical tweezers can stably trap GUVs containing iodixanol with controlled membrane tension. The trapped GUVs with high membrane tension can serve as a force sensor to accurately detect reversible folding of a DNA hairpin or membrane binding of synaptotagmin-1 C2AB domain attached to the GUV. We also observed that SUVs are rigid enough to resist large pulling forces and are suitable for detecting protein conformational changes induced by force. Our methodologies may facilitate single-molecule manipulation studies of membrane proteins using optical tweezers.     

A theoretical model of LDL-receptor trapping on a spherical cell.

The kinetics of the trapping of LDL-receptor complexes by coated pits on the surface of fibroblasts is examined in this paper. We have recently developed a mathematical formalism to extend Keizer's non-linear, non-equilibrium fluctuation-dissipation theory to the kinetics of chemical systems constrained to a spherical surface. Keizer's theory is ideally suited to the study of open biological systems. In the past it has been used to investigate endocytosis on fibroblasts. However, these applications have modeled the cell membrane with an infinite plane. As such, the finite size of the cellular membrane, as well as its precise symmetry, could not be incorporated into the previous studies. Thus in this paper we use our recently developed methodology to reexamine the trapping step in endocytosis on spherical cells. For cell surface processes, the theoretical consideration of a spherical symmetry or an infinite plane, in model calculations, will depend on the experimental or in vivo conditions of the processes of interest. For a spherical symmetry, we find that the finite size of the cell surface does not significantly affect the rate of the trapping step given the empirically determined values for the relevant parametes on fibroblasts. This result supports the approximation used in the previous investigation. However, this and other analyses indicate that the finitie size of the biological surface probably is an important parameter for processes which occur on smaller biological surfaces such as those found on organelles.     

The lysosomotropic agent monodansylcadaverine also acts as a solvent polarity probe.

The autofluorescent substance monodansylcadaverine has recently been reported as a specific in vivo marker for autophagic vacuoles. However, the mechanism for this specific labeling remained unclear. Our results reveal that the common model of ion trapping in acidic compartments cannot completely account for the observed autophagic vacuole staining. Because autophagic vacuoles are characterized by myelin-like membrane inclusions, we tested whether this lipid-rich environment is responsible for the staining properties of monodansylcadaverine. In in vitro experiments using either liposomes or solvents of different polarity, monodansylcadaverine showed an increased relative fluorescence intensity in a hydrophobic environment as well as a Stokes shift dependent on the solvent polarity. To test the effect of autophagic vacuoles or autophagic vacuole lipids on monodansylcadaverine fluorescence, we isolated autophagic vacuoles and purified autophagic vacuole lipids depleted of proteins. Entire autophagic vacuoles and autophagic vacuole lipids had the same effect on monodansylcadaverine fluorescence properties, suggesting lipids as the responsible component. Our results suggest that the in vivo fluorescence properties of monodansylcadaverine do not depend exclusively on accumulation in acidic compartments by ion trapping but also on an effective interaction of this molecule with autophagic vacuole membrane lipids. (J Histochem Cytochem 48:251-258, 2000)     

Differentiation between ligand trapping into intact cells and binding on muscarinic receptors

Binding properties of [3H] dexetimide , L-quinuclidinyl[phenyl-4-3H] benzilate and [3H]methylscopolamine were compared with intact 108 CC 15 cells and membrane preparations of those. The ability of the three ligands to label specifically muscarinic receptors on membrane fractions was quite similar. By contrast, when performed with intact cells, [3H] dexetimide and L-quinuclidinyl [phenyl-4-3H]benzilate revealed higher nonspecific binding which was prevented by methylamine, suggesting a trapping of the ligands within the cells presumably in the lysosomes. To the contrary, such nonspecific 'binding' or trapping was not detectable when [3H]methylscopolamine was used as ligand, a fact which makes this ligand particularly appropriate for labelling cell surface muscarinic receptors. It is concluded that more caution is needed in binding studies when performed with intact cells; indeed, besides specific binding on receptor sites, [3H]ligand can be entrapped within the cell and can even sometimes give the illusion of specific binding. The use of lysosomal agents which do not interfere with specific receptors on membrane preparations should allow one, in most cases, to discard the possibility of a trapping phenomenon in intact cells.     

The elimination mechanism of erythrocytes in vivo

The erythrocytes exhibit an unusual cyto-architecture. Because the cytosol is without organelles, the erythrocytes are cells, which have got only one single organelle, i.e. the plasmalemma. It reacts extremely sensitively to environmental factors with changes of the membrane structures, especially with an expression of IgG-and lectin receptor sites. This takes place during in vivo ageing, after attack of hydrolytic enzymes and after alteration of the membrane skeleton. The membrane bound IgG mediates the endocytosis of the macrophages (primary elimination). The role of the lectin receptors is unclear. The cell trapping (secondary elimination) is restricted to cells with extremely decreased deformability.     

Excitation trapping and charge separation in Photosystem II in the presence of an electrical field

To investigate the effects of a membrane potential on excitation trapping and charge separation in Photosystem II we have studied the chlorophyll fluorescence yield in osmotically swollen chloroplasts subjected to electrical field pulses. Significant effects were observed only in those membrane regions where a large membrane potential opposing the photochemical charge separation was built up. When the fluorescence yield was low, close to F₀, a much higher yield, up to F_max, was observed during the presence of the membrane potential. This is explained by an inhibition by the electrical field of electron transfer to the quinone acceptor Q, resulting in a decreased trapping of excitations. A field pulse applied when the fluorescence yield was high, Q and the donor side being in the reduced state, had the opposite effect: the fluorescence was quenched nearly to F₀. This field-induced fluorescence quenching is ascribed to reversed electron transfer from Q^? to the intermediate acceptor, pheophytin. Its field strength dependence suggests that the midpoint potential difference between pheophytin and Q is at most about 300 mV. Even then it must be assumed that electron transfer between pheophytin and Q spans 90% of the potential difference across the membrane.     

Excited state dynamics in photosystem I: effects of detergent and excitation wavelength.

Femtosecond transient absorption spectroscopy has been used to investigate the energy transfer and trapping processes in both intact membranes and purified detergent-isolated particles from a photosystem II deletion mutant of the cyanobacterium Synechocystis sp. PCC 6803, which contains only the photosystem I reaction center. Processes with similar lifetimes and spectra are observed in both the membrane fragments and the detergent-isolated particles, suggesting little disruption of the core antenna resulting from the detergent treatment. For the detergent-isolated particles, three different excitation wavelengths were used to excite different distributions of pigments in the spectrally heterogeneous core antenna. Only two lifetimes of 2.7-4.3 ps and 24-28 ps, and a nondecaying component are required to describe all the data. The 24-28 ps component is associated with trapping. The trapping process gives rise to a nondecaying spectrum that is due to oxidation of the primary electron donor. The lifetimes and spectra associated with trapping and radical pair formation are independent of excitation wavelength, suggesting that trapping proceeds from an equilibrated excited state. The 2.7-4.3 ps component characterizes the evolution from the initially excited distribution of pigments to the equilibrated excited state distribution. The spectrum associated with the 2.7-4.3 ps component is therefore strongly excitation wavelength dependent. Comparison of the difference spectra associated with the spectrally equilibrated state and the radical pair state suggests that the pigments in the photosystem I core antenna display some degree of excitonic coupling.     

Flux ratio theorems for nonlinear membrane transport under nonstationary conditions

We extend flux ratio theorems concerning ratios of unidirectional flux transients passed (in complementary experiments) through a medium of spatially inhomogeneous transport properties pertaining to diffusion, migration and temporary trapping of the transported substance. Any nonlinearity in the transport equations leads to a breakdown of the Ussing flux ratio theorem pertaining to all times. An integrated flux ratio theorem is proved for the case when the nonlinearity is in the kinetics of trapping, as when trapping sites can be saturated. The new theorem is shown to fail when the nonlinearity is due to a concentration-dependence of the diffusion coefficient, as in facilitated transport. The nature of a nonlinearity in membrane transport can therefore be elucidated experimentally by the use of the integrated flux ratio.     

Turning on ARF: the Sec7 family of guanine-nucleotide-exchange factors

ARF proteins are important regulators of membrane dynamics and protein transport within the eukaryotic cell. The Sec7 domain is approximately 200 amino acids in size and stimulates guanine-nucleotide exchange on members of the ARF class of small GTPases. The members of one subclass of Sec7-domain proteins are direct targets of the secretion-inhibiting drug brefeldin A, which blocks the exchange reaction by trapping a reaction intermediate in an inactive, abortive complex. A separate subclass of Sec7-domain proteins is involved in signal transduction and possess a domain that mediates membrane binding in response to extracellular signals.     

The sinusoidal domain of the plasma membrane of rat hepatocytes contains an amiloride-sensitive Na+/H+ antiport

ATP-dependent trapping of [14C]methylamine was demonstrated in vesicles selectively derived from the sinusoidal plasma membrane of rat hepatocytes; activity was lacking in vesicles prepared from the canalicular domain of the plasma membrane of rat hepatocytes. The proton movement was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, strophanthidin, vanadate, amiloride, and absence of sodium. 22Na efflux from sinusoidal membrane vesicles increased inversely to extravesicular pH. The results indicate that the sinusoidal plasma membrane of rat hepatocytes contains a Na+/H+ antiport.     

Nonequivalent nucleotide trapping in the two nucleotide binding folds of the human multidrug resistance protein MRP1

Multidrug resistance protein 1 (MRP1) and P-glycoprotein, which are ATP-dependent multidrug efflux pumps and involved in multidrug resistance of tumor cells, are members of the ATP binding cassette proteins and contain two nucleotide-binding folds (NBFs). P-glycoprotein hydrolyzes ATP at both NBFs, and vanadate-induced nucleotide trapping occurs at both NBFs. We examined vanadate-induced nucleotide trapping in MRP1 stably expressed in KB cell membrane by using 8-azido-[alpha-(32)P]ATP. Vanadate-induced nucleotide trapping in MRP1 was found to be stimulated by reduced glutathione, glutathione disulfide, and etoposide and to be synergistically stimulated by the presence of etoposide and either glutathione. These results suggest that glutathione and etoposide interact with MRP1 at different sites and that those bindings cooperatively stimulate the nucleotide trapping. Mild trypsin digestion of MRP1 revealed that vanadate-induced nucleotide trapping mainly occurs at NBF2. Our results suggest that the two NBFs of MRP1 might be functionally nonequivalent.     

Membrane translocation of lumenal domains of membrane proteins powered by downstream transmembrane sequences

Translocation of the N-terminus of a type I signal anchor (SA-I) sequence across the endoplasmic reticulum membrane can be arrested by tagging with a streptavidin-binding peptide tag (SBP tag) and trapping by streptavidin. In the present study, we first examine the affinity required for the translocation arrest. When the SBP tag is serially truncated, the ability for arrest gradually decreases. Surface plasmon resonance analysis shows that an interaction as strong as 10⁻⁸ M or a smaller dissociation constant is required for trapping the topogenesis of a natural SA-I sequence. Such truncated tags, however, become effective by mutating the SA-I sequence, suggesting that the translocation motivation is considerably influenced by the properties of the SA-I sequence. In addition, we introduce the SBP tag into lumenal loops of a multispanning membrane protein, human erythrocyte band 3. Among the tagged loops between transmembrane 1 (TM1) and TM8, three loops are trapped by cytosolic streptavidin. These loops are followed by TM sequences possessing topogenic properties, like the SA-I sequence, and translocation of one loop is diminished by insertion of a proline into the following TM sequence. These findings suggest that the translocation of lumenal loops by SA-I–like TM sequences has a crucial role in topogenesis of multispanning membrane proteins.     

Aminoglycosides versus bacteria – a description of the action, resistance mechanism, and nosocomial battleground

Since 1944, we have come a long way using aminoglycosides as antibiotics. Bacteria also have got them selected with hardier resistance mechanisms. Aminoglycosides are aminocyclitols that kill bacteria by inhibiting protein synthesis as they bind to the 16S rRNA and by disrupting the integrity of bacterial cell membrane. Aminoglycoside resistance mechanisms include: (a) the deactivation of aminoglycosides by N-acetylation, adenylylation or O-phosphorylation, (b) the reduction of the intracellular concentration of aminoglycosides by changes in outer membrane permeability, decreased inner membrane transport, active efflux, and drug trapping, (c) the alteration of the 30S ribosomal subunit target by mutation, and (d) methylation of the aminoglycoside binding site. There is an alarming increase in resistance outbreaks in hospital setting. Our review explores the molecular understanding of aminoglycoside action and resistance with an aim to minimize the spread of resistance.