Bacteriophage translocation

The occurrence of phages in the human body, especially in the gastrointestinal tract, raises the question of their potential role in the physiology and pathology of this system. Especially important is the issue of whether phages can pass the intestinal wall and migrate to lymph, peripheral blood, and internal organs and, if so, the effects such a phenomenon could have (such passage by bacteria, known as bacterial translocation, has been shown to cause various disturbances in humans, from immune defects to sepsis). Available data from the literature support the assumption that phage translocation can take place and may have some immunomodulatory effects. In addition, phages of the gut may play a protective role by inhibiting local immune reactions to antigens derived from gut flora.     

Chaperone-assisted translocation

We investigate the translocation of a stiff polymer through a nanopore in a membrane, in the presence of binding particles (chaperones) that bind reversibly to the polymer on both sides of the membrane. A bound chaperone covers one (univalent binding) or many (multivalent binding) binding sites. Assuming that the diffusion of the chaperones is fast compared to the rate of translocation we describe the process by a one-dimensional master equation. We expand previous models by a detailed study of the effective force in the master equation, which is obtained by the appropriate statistical mechanical average over the chaperone states. The dependence of the force on the degree of valency (the number of binding sites occupied by a chaperone) is studied in detail. We obtain finite size corrections (to the thermodynamical expression for the force), which, for univalent binding, can be expressed analytically. We finally investigate the mean velocity for translocation as a function of chaperone binding strength and size. For both univalent and multivalent binding simple results are obtained for the case of a sufficiently long translocating polymer.     

Turgor-regulated translocation

Abstract. The role played by potassium in plants is examined, in particular its connection with phloem translocation. The possibility emerges that trans-location may be a turgor-regulated process in which potassium plays a central role.
A simple mechanistic hypothesis is proposed and the evidence for it is discussed. The hypothesis is strongly supported by both the direct and the circumstantial evidence.
An important conclusion is the need to reassess the importance of the translocation process in the control of assimilate partitioning. Hitherto, as implied by Münch's hypothesis, control has been assumed to be exerted by source and/or sink activity with the translocation pathway playing a more passive secondary role. If the present proposal is correct the translocation process emerges instead as a dominant factor in the control of assimilate partitioning.     

Studies of translocation catalysis

There is a symbiotic relationship between the evolution of fundamental theory and the winning of experimentally-based knowledge. The impact of the General Chemiosmotic Theory on our understanding of the nature of membrane transport processes is described and discussed. The history of experimental studies on transport catalysed by ionophore antibiotics and the membrane proteins of mitochondria and bacteria are used to illustrate the evolution of knowledge and theory. Recent experimental approaches to understanding the lactose-H⁺ symport protein ofEscherichia coli and other sugar porters are described to show that the lack of experimental knowledge of the three-dimensional structures of the proteins currently limits the development of theories about their molecular mechanism of translocation catalysis.     

Apetiolar photosynthate translocation

Apetiolar transport of photosynthate ⁻¹⁴C has been studied by feeding of ¹⁴CO₂ to soybean petioles. Translocation occurs in the absence of leaves, but both the rate and velocity are diminished. The effect of root excision is not as profound as that of leaves. It appears, in some instances, to inhibit transport partially, so that accumulation of photosynthate develops, giving a steeper isotopic gradient. The effect of leaf darkening is to diminish its uptake of photosynthate from the petiole, possibly as a result of decreased transpiration in the lowered temperature of the darkened leaf. The data suggest that neither mass flow nor active transport provide an adequate basis for normal photosynthate transport but that the leaves provide a direct force requiring structural continuity, or a translocation carrier.     

204 Polymer translocation

The translocation of a single macromolecule through a protein pore or a solid-state nanopore involves three major stages: (1) approach of the macromolecule towards the pore, (2) capture/recognition of the macromolecule at the pore entrance, and (3) threading through the pore (see the Figure) (Muthukumar, 2011). All of these stages are controlled by conformational entropy of the macromolecule, charge decoration, and the geometry of the pore, hydrodynamics, and electrostatic interactions. Chief among the contributing factors are the entropic barrier presented by the pore to the penetration of the macromolecule, pore–polymer interactions, electro-osmotic flow, and the drift-diffusion of the macromolecule in electrolyte solutions. A unifying theory of these contributing factors will be described in the context of a few illustrative experimental data on DNA translocation and protein translocation through protein pores and solid-state nanopores. Future challenges to specific biological systems will be briefly discussed.     

生物大分子的细胞核质转运

生物大分子通过细胞核孔复合体的转运是真核细胞基因复制、转录和翻译的必要环节 ,也是联系细胞核内外信号转递与参与细胞内核反应 (即细胞增殖、分化、凋亡等核反应 )调控的重要环节。本文主要介绍细胞核孔复合体结构、出入细胞核的转运过程及核转运蛋白与亲核素方面的研究进展 ,细胞核转运过程的深入研究在医药学基础和临床实践都有十分重要的意义。     

Nanoelectropulse-induced phosphatidylserine translocation

Nanosecond, megavolt-per-meter, pulsed electric fields induce phosphatidylserine (PS) externalization, intracellular calcium redistribution, and apoptosis in Jurkat T-lymphoblasts, without causing immediately apparent physical damage to the cells. Intracellular calcium mobilization occurs within milliseconds of pulse exposure, and membrane phospholipid translocation is observed within minutes. Pulsed cells maintain cytoplasmic membrane integrity, blocking propidium iodide and Trypan blue. Indicators of apoptosis-caspase activation and loss of mitochondrial membrane potential-appear in nanoelectropulsed cells at later times. Although a theoretical framework has been established, specific mechanisms through which external nanosecond pulsed electric fields trigger intracellular responses in actively growing cells have not yet been experimentally characterized. This report focuses on the membrane phospholipid rearrangement that appears after ultrashort pulse exposure. We present evidence that the minimum field strength required for PS externalization in actively metabolizing Jurkat cells with 7-ns pulses produces transmembrane potentials associated with increased membrane conductance when pulse widths are microseconds rather than nanoseconds. We also show that nanoelectropulse trains delivered at repetition rates from 2 to 2000 Hz have similar effects, that nanoelectropulse-induced PS externalization does not require calcium in the external medium, and that the pulse regimens used in these experiments do not cause significant intra- or extracellular Joule heating.     

Models of post-translational protein translocation

Organellar Hsp-70 is required for post-translational translocation into the endoplasmic reticulum and mitochondria. The functional role played by Hsp-70 is unknown. However, two operating principles have been suggested. The power stroke model proposes that Hsp-70 undergoes a conformational change, which pulls the precursor protein through the translocation pore, whereas, in the Brownian ratchet model, the role of Hsp-70 is simply to block backsliding through the pore. A mathematical analysis of both mechanisms is presented and reveals that qualitative differences between the models occur in the behavior of the mean velocity and effective diffusion coefficient as a function of Hsp-70 concentration. An experimental method is proposed for measuring these two quantities that only relies on current experimental techniques.     

小麦——黑麦染色体易位系的细胞学鉴定

用C－带技术分析了普通小麦“中国春”、黑麦“胜利”及小黑麦与普通小麦经辐射处理的后代中产生的并经多代纯化的5个带有黑麦某些性状的普通小麦品系的根尖染色体,结果表明：品系98－5－1为1A／1R纯合易位系,具有抗锈病、抗白粉病等基因,可作为诱导小片段易位的资源。作者提出在小麦－黑麦易位系鉴定中应用更高分辩率的G－带技术识别黑麦染色体片段或小片段易位。     

Enzymatic detection of protein translocation

Fundamental to eukaryotic cell signaling is the regulation of protein function by directed localization. Detection of these events has been largely qualitative owing to the limitations of existing technologies. Here we describe a method for quantitatively assessing protein translocation using proximity-induced enzyme complementation. The complementation assay for protein translocation (CAPT) is derived from beta-galactosidase and comprises one enzyme fragment, omega, which is localized to a particular subcellular region, and a small complementing peptide, alpha, which is fused to the protein of interest. The concentration of alpha in the immediate vicinity of omega correlates with the amount of enzyme activity obtained in a dose- and time-dependent manner, thus acting as a genetically encoded biosensor for local protein concentration. Using CAPT, inducible protein movement from the cytosol to the nucleus or plasma membrane was quantitatively monitored in multiwell format and in live mammalian cells by flow cytometry.     

Twin-arginine-dependent translocation of folded proteins

Twin-arginine translocation (Tat) denotes a protein transport pathway in bacteria, archaea and plant chloroplasts, which is specific for precursor proteins harbouring a characteristic twin-arginine pair in their signal sequences. Many Tat substrates receive cofactors and fold prior to translocation. For a subset of them, proofreading chaperones coordinate maturation and membrane-targeting. Tat translocases comprise two kinds of membrane proteins, a hexahelical TatC-type protein and one or two members of the single-spanning TatA protein family, called TatA and TatB. TatC- and TatA-type proteins form homo- and hetero-oligomeric complexes. The subunits of TatABC translocases are predominantly recovered from two separate complexes, a TatBC complex that might contain some TatA, and a homomeric TatA complex. TatB and TatC coordinately recognize twin-arginine signal peptides and accommodate them in membrane-embedded binding pockets. Advanced binding of the signal sequence to the Tat translocase requires the proton-motive force (PMF) across the membranes and might involve a first recruitment of TatA. When targeted in this manner, folded twin-arginine precursors induce homo-oligomerization of TatB and TatA. Ultimately, this leads to the formation of a transmembrane protein conduit that possibly consists of a pore-like TatA structure. The translocation step again is dependent on the PMF.     

Membrane translocation of folded proteins

An ever-increasing number of proteins have been shown to translocate across various membranes of bacterial as well as eukaryotic cells in their folded states as a part of physiological and/or pathophysiological processes. Herein, we provide an overview of the systems/processes that are established or likely to involve the membrane translocation of folded proteins, such as protein export by the twin-arginine translocation system in bacteria and chloroplasts, unconventional protein secretion and protein import into the peroxisome in eukaryotes, and the cytosolic entry of proteins (e.g., bacterial toxins) and viruses into eukaryotes. We also discuss the various mechanistic models that have previously been proposed for the membrane translocation of folded proteins including pore/channel formation, local membrane disruption, membrane thinning, and transport by membrane vesicles. Finally, we introduce a newly discovered vesicular transport mechanism, vesicle budding and collapse, and present evidence that vesicle budding and collapse may represent a unifying mechanism that drives some (and potentially all) of folded protein translocation processes.     

The translocation mechanism of P-glycoprotein

Multidrug transporters are involved in mediating the failure of chemotherapy in treating several serious diseases. The archetypal multidrug transporter P-glycoprotein (P-gp) confers resistance to a large number of chemically and functionally unrelated anti-cancer drugs by mediating efflux from cancer cells. The ability to efflux such a large number of drugs remains a biological enigma and the lack of mechanistic understanding of the translocation pathway used by P-gp prevents rational design of compounds to inhibit its function. The translocation pathway is critically dependent on ATP hydrolysis and drug interaction with P-gp is possible at one of a multitude of allosterically linked binding sites. However, aspects such as coupling stoichiometry, molecular properties of binding sites and the nature of conformational changes remain unresolved or the centre of considerable controversy. The present review attempts to utilise the available data to generate a detailed sequence of events in the translocation pathway for this dexterous protein.     

Pre-steady-state kinetics of ribosomal translocation

The two partial reactions of elongation factor G dependent translocation, the release of deacylated tRNA from the P site and the displacement of peptidyl tRNA from the A to the P site, have been studied with the stopped-flow technique. The experiments were performed with poly(U)-programmed ribosomes from Escherichia coli carrying deacylated tRNAPhe in the P site and N-AcPhe-tRNAPhe in the A site in the presence of GTP. The kinetics of the reaction were followed by monitoring either the intensity or the polarization of the fluorescence of both wybutine and proflavine located in the anticodon loop or of proflavine located in the D loop of yeast tRNAPhe or N-AcPhe-tRNAPhe. Both displacement and release fluorescence changes could be described by three exponentials, exhibiting apparent first-order rate-constants (20 degrees C) of 2 to 5 s-1 (15 s-1, 35 degrees C), 0.1 to 0.3 s-1, and 0.01 to 0.02 s-1, measured with a saturating concentration of elongation factor G (1 microM). The activation energy for the fast process of both reactions was found to be 70 kJ/mol (17 kcal/mol), while the intermediate process exhibits an activation energy of 30 kJ/mol (7 kcal/mol). The fast step is assigned to the displacement of the N-AcPhe-tRNAPhe from the A to the P site, and to the release of the tRNAPhe from the P site. The reactions take place simultaneously to form an intermediate post-translocation complex. The latter, in the intermediate step, rearranges to form a post-translocation complex carrying the deacylated tRNAPhe in an exit site and N-AcPhe-tRNAPhe in the P site, both in their equilibrium states. In parallel, or subsequently, the deacylated tRNAPhe spontaneously dissociates from the ribosome, thus completing the translocation process. The slow process has not been assigned.