Modes Of Translocation Research Articles

Protein Targeting: Entropy, Energetics and Modular Machines

New light is being shed on the mechanism of protein import into mitochondria. The inner membrane translocase can switch between modes of translocation, and assists what might be an entropic device to drive the initial entry of substrate proteins across the outer membrane.

A Little Help from My Friends: Quality Control of Presecretory Proteins in Bacteria

All organisms, including bacteria, localize a fraction of all of their proteins partially or completely outside of the cytosol. Along the way, these proteins must cross at least one hydrophobic lipid membrane. The remarkable feat of delivering proteins across tightly sealed membranes is achieved largely by complex secretion machineries known as translocons. These machines recognize their substrates via signal sequences, which are required for proper targeting to the translocon. The bulk of protein transport across the inner cytoplasmic membrane is facilitated by the well-known general secretory (Sec) pathway, but additional categories for transport into or across the inner membrane, including the recently discovered twin-arginine translocation (Tat) pathway, exist. More-specialized mechanisms for targeting proteins to the inner membrane, the outer membrane, or the extracellular environment also exist and are reviewed elsewhere (19, 31, 71). Proteins targeted to the Sec pathway achieve membrane translocation through the Sec translocon, a proteinaceous conduit formed by an oligomeric assembly of the heterotrimeric membrane protein complex SecYEG (7, 79) and the peripheral ATPase SecA as a molecular motor (26). Sec substrates traverse the membrane in a largely unfolded state and effectively thread their way through the pore. In stark contrast to the Sec-dependent threading of unstructured substrates, the Tat pathway has the unique ability to transport proteins that have attained a substantial degree of tertiary or even quaternary structure in the cytoplasm prior to membrane translocation (13, 22, 35, 66, 70). This process is enabled by a translocon consisting of the TatA, TatB, TatC, and TatE proteins, which share little homology with the components of the Sec translocon. Consistent with these distinct modes of translocation, both the Sec and Tat pathways have evolved unique measures for surveying the quality of their respective substrates. This minireview will discuss how the proper structural integrity of proteins to be transported (hereinafter referred to as preproteins) is ensured during the early stages of Sec and Tat targeting so that these proteins remain compatible with their respective macromolecular transport machineries.

Multipolar Migration: The Third Mode of Radial Neuronal Migration in the Developing Cerebral Cortex

Two distinct modes of radial neuronal migration, locomotion and somal translocation, have been reported in the developing cerebral cortex. Although these two modes of migration have been well documented, the cortical intermediate zone contains abundant multipolar cells, and they do not resemble the cells migrating by locomotion or somal translocation. Here, we report that these multipolar cells express neuronal markers and extend multiple thin processes in various directions independently of the radial glial fibers. Time-lapse analysis of living slices revealed that the multipolar cells do not have any fixed cell polarity, and that they very dynamically extend and retract multiple processes as their cell bodies slowly move. They do not usually move straight toward the pial surface during their radial migration, but instead frequently change migration direction and rate; sometimes they even remain in almost the same position, especially when they are in the subventricular zone. Occasionally, the multipolar cells jump tangentially during their radial migration. Because the migration modality of these cells clearly differs from locomotion or somal translocation, we refer to their novel type of migration as "multipolar migration." In view of the high proportion of cells exhibiting multipolar migration, this third mode of radial migration must be an important type of migration in the developing cortex.

Characterization of a lactose permease mutant that binds IIAGlc in the absence of ligand.

Enzyme IIA(Glc) of the Escherichia coli phosphoenolpyruvate:glucose phosphotransferase system plays a direct role in regulating inducible transport systems. Dephosphorylated IIA(Glc) binds directly to lactose permease in a reaction that requires binding of a galactosidic substrate. A double-Cys mutation (Ile129 --> Cys/Lys131 --> Cys) was introduced into helix IV of the permease near the IIA(Glc) binding site in cytoplasmic loop IV/V and in the vicinity of the galactoside binding site at the interface of helices IV, V, and VIII. The mutant no longer requires galactoside for IIA(Glc) binding as demonstrated by both a [(125)I]IIA(Glc) binding assay and a newly developed fluorescence anisotropy assay. Further characterization of the mutant shows that it binds substrate with high affinity, but is almost completely defective in all modes of translocation across the cytoplasmic membrane. The data are consistent with the interpretation that the double mutant is locked in an inward-facing conformation.

Neuronal migration in the developing cerebral cortex: observations based on real-time imaging.

We have used time-lapse imaging of acute cortical slices to study the migration of neurons from their sites of origin to their positions in the developing neocortex. We found that two distinct modes of cell movement, somal translocation and glia-guided locomotion, are responsible for the radial migration of neurons generated in the cortical ventricular zone. The former is the prevalent form of radial movement of the early-born cortical neurons, while the latter is adopted by those generated later in corticogenesis. Interneurons, found to originate in the ganglionic eminence, follow tangential migratory paths to reach the developing cortex. Upon reaching the cortex, these cells seek the ventricular zone using a mode of movement that we have termed 'ventricle-directed migration', before they migrate to their positions in the cortical plate. In addition to these forms of movement, we report here a unique morphological and migratory behavior for a population of cortical neurons. These cells are multipolar in form, and are highly motile in the formation and retraction of their processes. Based on these morphological features, we refer to this type of cells as 'branching cells' and attribute the phenotype to a subset of cortical interneurons.

Pharmacokinetics of sparfloxacin in the serum and vitreous humor of rabbits: physicochemical properties that regulate penetration of quinolone antimicrobials.

We have used a recently described animal model to characterize the ocular pharmacokinetics of sparfloxacin in vitreous humor of uninfected albino rabbits following systemic administration and direct intraocular injection. The relationships of lipophilicity, protein binding, and molecular weight to the penetration and elimination of sparfloxacin were compared to those of ciprofloxacin, fleroxacin, and ofloxacin. To determine whether elimination was active, elimination rates following direct injection with and without probenecid or heat-killed bacteria were compared. Sparfloxacin concentrations were measured in the serum and vitreous humor by a biological assay. Protein binding and lipophilicity were determined, respectively, by ultrafiltration and oil-water partitioning. Pharmacokinetic parameters were characterized with RSTRIP, an iterative, nonlinear, weighted, least-squares-regression program. The relationship between each independent variable and mean quinolone concentration or elimination rate in the vitreous humor was determined by multiple linear regression. The mean concentration of sparfloxacin in the vitreous humor was 59.4% +/- 12.2% of that in serum. Penetration of sparfloxacin, ciprofloxacin, fleroxacin, and ofloxacin into, and elimination from, the vitreous humor correlated with lipophilicity (r2 > 0.999). The linear-regression equation describing this relationship was not improved by including the inverse of the square root of the molecular weight and/or the degree of protein binding. Elimination rates for each quinolone were decreased by the intraocular administration of probenecid. Heat-killed Staphylococcus epidermidis decreased the rate of elimination of fleroxacin. Penetration of sparfloxacin into the noninflamed vitreous humor was greater than that of any quinolone previously examined. There was an excellent correlation between lipophilicity and vitreous entry or elimination for sparfloxacin as well as ciprofloxacin, fleroxacin, and ofloxacin. There are two modes of quinolone translocation into and out of the vitreous humor: diffusion into the eye and both diffusion and carrier-mediated elimination out of the vitreous humor.

Chloride and proton transport in bacteriorhodopsin mutant D85T: different modes of ion translocation in a retinal protein

Replacement of aspartate 85 (D85) in bacteriorhodopsin (BR) by threonine but not be asparagine creates at pH<7 an anion-binding site in the molecular similar to that in chloride pump halorhodopsin. Binding of various anions to BR-D85T causes a blue shift of the absorption maximum by maximally 57 nm. Connected to this color change is a change in the absorption difference spectrum of the initial state and the longest living photo intermediate from a positive difference maximum at 460 nm in the absence of transported anions to one at 630 nm in their presence. Increasing anion concentration cause decreasing decay times of this intermediate. At physiological pH, BR-D85T but not BR-D85N transports chloride ions inward in green light, protons outward in blue or green light and protons inward in white light (directions refer to the intact cell). The proton movements are observable also in BR-D85N. Thus, creation of an anion-binding site in BR is responsible for chloride transport and introduction of anion-dependent spectroscopic properties at physiological pH. The different transport modes are explained with the help of the recently proposed IST model, which states that after light-induced isomerization of the retinal an ion transfer step and an accessibility change of the active site follow. The latter two steps occur independently. In order to complete the cyclic event, the accessibility change, ion transfer and isomerization state have to be reversed. The relative rates of accessibility changes and ion transfer steps define ultimately the vectoriality of ion transfers. All transport modes described here for the same molecule can satisfactorily be described in the framework of this general concept.

Different modes of proton translocation by sensory rhodopsin I.

The membrane-bound complex between sensory rhodopsin I (SRI) and its transducer HtrI forms the functional photoreceptor unit that allows transmission of light signals to the flagellar motor. Although being a photosensor, SRI, the mutant SRI-D76N and the HtrI-SRI complex can transport protons, as we demonstrate by using the sensitive and ion-specific black lipid membrane technique. SRI sustains an orange light-driven (one-photon-driven) outward proton transport which is enhanced by additional blue light (two-photon-driven). The vectoriality of the two-photon-driven transport could be reversed at neutral pH from the outward to the inward direction by switching the cut-off wavelength of the long wavelength light from 550 to 630 nm. The cut-off wavelength determining the reversal point decreases with decreasing pH. The currents could be enhanced by azide. A two-photon-driven inward proton transport by SRI-D76N (catalyzed by azide) and by the complex HtrI-SRI is demonstrated. The influence of pH and azide concentration on the rise and decay kinetics of the SRI380 intermediate is analyzed. The different modes of proton translocation of the SRI species are discussed on the basis of a general model of proton translocation of retinal proteins and in the context of signal transduction.

Structure and Function of Halorhodopsin

AbstractA comprehensive review of the physiology, structure, and function of halorhodopsin (HR), the only known light‐driven anion pump, is presented. Beside the well‐studied transport function of HR in intact cells the article focuses on recent results about the molecular properties of HR. Overexpression and in vivo 2‐D crystallization allowed structural investigations at a level of 7 Å resolution. The results demonstrate a very close structural relationship with the proton pump bacteriorhododopsin (BR). The retinal binding site as well as a chloride binding site near the Schiff base in HR can be modeled with the side chains placed into the corresponding positions in the BR structural model. Mechanistically the vectorial catalytic cycle of HR is similar to that of BR, as suggested before (Oesterhelt et al., J. Bioenerg. Biomembr. 1992, 24: 181), and consists of photoisomerization of the retinal moiety which triggers chloride movement within the transport site towards the Schiff base. This is followed by an accessibility change from the extracellular (EC) channel to the cytoplasmic (CP) channel allowing chloride to be released into the cytoplasm. After reisomerization and reversion of the accessibility change, a chloride is rebound into the transport site from the extracellular surface through EC. In the absence of transported ions or the additional presence of azide, photoisomerization to 13‐cis is followed directly by an accessibility change and release of a proton from the Schiff base through CP. Blue light causes photoisomerization back to trans and the accessibility change is reversed. Uptake of a proton through EC completes proton transport from the outside to the inside. Depending on relative concentrations of chloride and azide, both modes of ion translocation operate in parallel and as alternatives during individual cycles of a molecule. Future experimentation will have to fill in the many details of this molecular model of ion transport.

A large presecretory protein translocates both cotranslationally, using signal recognition particle and ribosome, and post-translationally, without these ribonucleoparticles, when synthesized in the presence of mammalian microsomes.

Translocation of large presecretory proteins into the mammalian endoplasmic reticulum requires the ribonucleoparticles, signal recognition particle, and ribosome and is tightly coupled to ongoing protein synthesis. We have shown previously that small presecretory proteins can translocate post-translationally in a reaction that does not require these ribonucleoparticles. We now report that one large protein, a synthetic hybrid between preprocecropin A and dihydrofolate reductase, translocates both cotranslationally (with the aid of signal recognition particle and ribosome) and post-translationally (without the involvement of these ribonucleoparticles) during its in vitro synthesis in the presence of dog pancreas microsomes. The distinction between these two modes of translocation was made possible by adding methotrexate to the translocation reaction. Methotrexate can only form a tight complex with those preprocecropin A-dihydrofolate reductase hybrid chains that have completed their synthesis and folded, but in forming this tight complex, this drug prevents translocation of the dihydrofolate reductase domain across the membrane.

Site-directed mutagenesis of tyrosine residues in the lac permease of Escherichia coli.

By using oligonucleotide-directed, site-specific mutagenesis, each of the 14 Tyr residues in the lac permease of Escherichia coli was replaced with Phe, and the activity of each mutant was studied with respect to active transport, equilibrium exchange, and efflux. Ten of the mutations have no significant effect on permease activity. Of the four mutations that alter activity, replacement of Tyr26 or Tyr336 with Phe severely decreases all modes of translocation, and the binding affinity of the mutant permease for p-nitrophenyl alpha-D-galactopyranoside is markedly decreased (i.e., KD is increased). In addition, the Phe336 mutant permease is inserted into the membrane to a lesser extent than wild-type permease, as judged by immunoblot experiments. Permease containing Phe in place of Tyr236 catalyzes lactose exchange approximately 40% as well as wild-type permease but does not catalyze active transport or efflux. Finally, permease with Phe in place of Tyr382 catalyzes equilibrium exchange normally, but exhibits low rates of active transport and efflux without being uncoupled, thereby suggesting that replacement of Tyr382 with Phe alters a kinetic step involving translocation of the unloaded permease across the membrane.

Effect of distance and orientation between arginine-302, histidine-322, and glutamate-325 on the activity of lac permease from Escherichia coli

lac permease of Escherichia coli was modified by site-directed mutagenesis in order to investigate the effects of polarity, distance, and orientation between the components of a putative H+ relay system (Arg302/His322/Glu325) postulated to be involved in lactose-coupled H+ translocation. The importance of polarity between His322 and Glu325 was studied by interchanging the residues, and the modified permease--H322E/E325H--is inactive in all modes of translocation. The effect of distance and/or orientation between His322 and Glu325 was investigated by interchanging Glu325 with Val326, thereby moving the carboxylate one residue around putative helix X. The resulting permease molecule--E325V/V326E--is also completely inactive; control mutations, E325V [Carrasco, N., Püttner, I. B., Antes, L. M., Lee, J. A., Larigan, J. D., Lolkema, J. S., Roepe, P. D., & Kaback, H. R. (1989) Biochemistry (second paper of three in this issue)], and E325A/V326E, indicate that a Glu residue at position 326 inactivates the permease. The wild-type orientation between His and Glu was then restored by further mutation of E325V/V326E to introduce a His residue into position 323 or by interchanging Met323 with His322. The resulting permease molecules--M323H/E325V/V326E and H322M/M323H/E325V/V326E--contain the wild-type His/Glu orientation, but the His/Glu ion pair is rotated about the helical axis by 100 degrees relative to Arg302 in putative helix IX. Both mutants are inactive with respect to all modes of translocation. The results provide strong support for the contention that the polarity between His322 and Glu325 and the geometric relationship between Arg302, His322, and Glu325 are critical for permease activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of site-directed mutants in the lac permease of Escherichia coli. 1. Replacement of histidine residues.

Wild-type lac permease from Escherichia coli and two site-directed mutant permeases containing Arg in place of His35 and His39 or His322 were purified and reconstituted into proteoliposomes. H35-39R permease is indistinguishable from wild type with regard to all modes of translocation. In contrast, purified, reconstituted permease with Arg in place of His322 is defective in active transport, efflux, equilibrium exchange, and counterflow but catalyzes downhill influx of lactose without concomitant H+ translocation. Although permease with Arg in place of His205 was thought to be devoid of activity [Padan, E., Sarkar, H. K., Viitanen, P. V., Poonian, M. S., & Kaback, H. R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6765], sequencing of lac Y in pH205R reveals the presence of two additional mutations in the 5' end of the gene, and replacement of this portion of lac Y with a restriction fragment from the wild-type gene yields permease with normal activity. Permeases with Asn, Gln, or Lys in place of His322, like H322R permease, catalyze downhill influx of lactose without H+ translocation but are unable to catalyze active transport, equilibrium exchange, or counterflow. Unlike H322R permease, however, the latter mutants catalyze efflux at rates comparable to that of wild-type permease, although the reaction does not occur in symport with H+. Finally, as evidenced by flow dialysis and photoaffinity labeling experiments, replacement of His322 appears to cause a marked decrease in the affinity of the permease for substrate. The results confirm and extend the contention that His322 is the only His residue in the permease involved in lactose/H+ symport and that an imidazole moiety at position 322 is obligatory.(ABSTRACT TRUNCATED AT 250 WORDS)

Modes of proton translocation across the cell membrane of respiring cyanobacteria

Acidification of weakly buffered suspensions of the cyanobacteria Anacystis nidulans, Nostoc sp. strain MAC, Dermocarpa sp. and Anabaena variabilis was observed after the application of oxygen pulses to anaerobic cells. The acidification was caused by proton extrusion from the oxygen pulsed cells since it was eliminated by the uncoupler (H+ ionophore) carbonyl cyanide m-chlorophenylhydrazone. Results with the inhibitors dicyclohexylcarbodiimide or 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, orthovanadate and cyanide indicated the association of various fractions of the observed proton extrusion with different activities of the cell membrane, viz. a H+-translocating reversible F0F1-ATPase, a unidirectional H+-translocating ATP hydrolase, and a respiratory electron transport system, respectively. Further parameters investigated were the pH dependence and the H+/O stoichiometry of the H+ extrusion from oxygen pulsed cyanobacteria. H+/O ratios at neutral pH were between 4 (Anacystis nidulans) and 0.3 (Dermocarpa) with uninhibited, actively phosphorylating cells and between 2 (Anacystis nidulans) and 0.4 (Dermocarpa) with ATPase-inhibited (ATP-depleted) cells, respectively. It is significant that with all four cyanobacteria tested a major fraction of the observed H+ ejection remained unaffected by ATPase inhibitors even at concentration which completely abolished all oxidative phosphorylation. Vanadate had a major effect on the H+ extrusion from Anabaena only. From this it is concluded that in the cyanobacterial species investigated part of the H+ extrusion from oxygen pulsed cells is directly linked to some H+-translocating respiratory electron transport chain present in the cell membrane.

52] Control of intracellular protein traffic

Publisher Summary This chapter describes the control of intracellular protein traffic including translocation of proteins across membranes, mechanisms of translocation, integration into membranes, phylogeny of membranes, protein translocation and compartments, and posttranslocational pathways. The collective term topogenesis refers to encompass protein translocation (partial or complete) across membranes, and subsequent post-translocational protein traffic. Two modes of translocation are distinguished—cotranslational and posttranslational modes. In cotranslational translocation, the passage of the polypeptide chain across the membrane appears to be strictly coupled to translation, whereas in posttranslational translocation the polypeptide can traverse the membrane post-translationally uncoupled from its synthesis. Translocation is not always accompanied by cleavage of the signal sequence, and there are numerous examples for uncleaved signal sequences. The chapter discusses two approaches to dissect the membrane translocation activity: salt extraction and limited proteolysis. However, many integral membrane proteins (IMPs) require selective translocation of one or more hydrophilic segment(s) of the polypeptide chain to acquire their characteristic asymmetric orientation.

Binding of adenosine diphosphate and of antagonist ligands to the mitochondrial ADP carrier

The properties of adenine nucleotide translocation in mitochondria are reviewed. The inhibitory properties of specific inhibitors: (35S)-atractyloside, (35S)carboxyatractyloside (or (35S)gummiferin), (14C)bongkrekic acid, are analysed and compared to their binding properties. In contrast to atractyloside, carboxyatractyloside is not a competitive inhibitor of ADP binding and ADP translocation. Double labelling experiments using (35S)-atractyloside and (3H)ADP confirm that ADP and ATR compete for binding to the inner mitochondrial membrane. Binding of ADP to its translocase brings about membrane conformation changes with the exposure of a small number of -SH groups. Some models illustrating possible modes of translocation which incorporate these findings are discussed. Après une revue des propriétés physico-chimiques de la translocation des adénine nucléotides dans les mitochondries, nous avons analysé les propriétés d'inhibition et de fixation d'inhibiteurs spécifiques: (35S)-atractyloside, (35S)carboxyatractyloside (ou (35S)gummiferine) et acide (14C)bongkrekique. Le carboxyatractyloside contrairement à l'atractyloside n'est pas un inhibiteur compétitif de la fixation et de la translocation d'ADP. Des expériences de double marquage mettant en œuvre du (35S)atractyloside et du (3H)-ADP corroborent la compétition entre ADP et atractyloside pour leur fixation au niveau de la membrane interne mitochondriale. La fixation de l'ADP sur son transporteur spécifique induit un changement de conformation membranaire qui se traduit en particulier par un démasquage d'un nombre limité de groupes -SH. Différents modèles de transport d'ADP sont discutés en fonction des résultats expérimentaux.