Translocation of proteins across archaeal cytoplasmic membranes

All cells need to transport proteins across hydrophobic membranes. Several mechanisms have evolved to facilitate this transport, including: (i) the universally-conserved Sec system, which transports proteins in an unfolded conformation and is thought to be the major translocation pathway in most organisms and (ii) the Tat system, which transports proteins that have already obtained some degree of tertiary structure. Here, we present the current understanding of these processes in the domain Archaea, and how they compare to the corresponding pathways in bacteria and eukaryotes.     

Translocation of proteins across the multiple membranes of complex plastids.

Secondary endosymbiosis describes the origin of plastids in several major algal groups such as dinoflagellates, euglenoids, heterokonts, haptophytes, cryptomonads, chlorarachniophytes and parasites such as apicomplexa. An integral part of secondary endosymbiosis has been the transfer of genes for plastid proteins from the endosymbiont to the host nucleus. Targeting of the encoded proteins back to the plastid from their new site of synthesis in the host involves targeting across the multiple membranes surrounding these complex plastids. Although this process shows many overall similarities in the different algal groups, it is emerging that differences exist in the mechanisms adopted.     

Translocation of nascent secretory proteins across membranes can occur late in translation.

Signal recognition particle (SRP) causes an arrest in the translation of nascent secretory proteins in a wheat germ cell-free system. In order to examine at what point during the synthesis of a secretory protein its translocation across the endoplasmic reticulum (ER) membrane can occur, SRP was used to arrest nascent chain elongation at various times during a synchronous translation, thus allowing the generation of nascent chains of increasing length. It was found that SRP can still bring about an arrest as late as when an average of two-thirds of nascent IgG light chain was completed. Rough microsomes were added to translations blocked with SRP to determine if such relatively long nascent chains could still be translocated across the membrane. It was found that nascent chains which had been arrested by SRP, regardless of their length, could be translocated into rough microsomes. In the case of IgG light chain, translocation levels of 50% were still observed with nascent chains corresponding to as much as 70-75% of the intact preprotein. Similar results were observed for the nascent bovine prolactin precursor. These results demonstrate that the synthesis of secretory proteins can be uncoupled from their translocation, and that fairly large nascent chains are capable of crossing the membrane of the ER post-translationally.     

Translocation of histone proteins across lipid bilayers and Mycoplasma membranes

We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).     

Translocation of a phycoerythrin alpha subunit across five biological membranes

Cryptophytes, unicellular algae, evolved by secondary endosymbiosis and contain plastids surrounded by four membranes. In contrast to cyanobacteria and red algae, their phycobiliproteins do not assemble into phycobilisomes and are located within the thylakoid lumen instead of the stroma. We identified two gene families encoding phycoerythrin alpha and light-harvesting complex proteins from an expressed sequence tag library of the cryptophyte Guillardia theta. The proteins bear a bipartite topogenic signal responsible for the transport of nuclear encoded proteins via the ER into the plastid. Analysis of the phycoerythrin alpha sequences revealed that more than half of them carry an additional, third topogenic signal comprising a twin arginine motif, which is indicative of Tat (twin arginine transport)-specific targeting signals. We performed import studies with several derivatives of one member using a diatom transformation system, as well as intact chloroplasts and thylakoid vesicles isolated from pea. We demonstrated the different targeting properties of each individual part of the tripartite leader and show that phycoerythrin alpha is transported across the thylakoid membrane into the thylakoid lumen and protease-protected. Furthermore, we showed that thylakoid transport of phycoerythrin alpha takes place by the Tat pathway even if the 36 amino acid long bipartite topogenic signal precedes the actual twin arginine signal. This is the first experimental evidence of a protein being targeted across five biological membranes.     

Translocation of polysialic acid across model membranes: kinetic analysis and dynamic studies.

Transmembrane translocation of polyion homopolymers takes place in the case of polyanionic polysialic acid (polySia), polyanionic polynucleotides and polycationic polypeptides. The purpose of this work was to determine the role of membrane electrical parameters on the kinetics of polyion translocation, the influence of polysialic acid on ion adsorption on positively charged membrane surface and the dynamics of the phospholipid hydrocarbon chains and choline group by using 1H-NMR. The analysis of polyion translocation was performed by using the electrical equivalent circuit of the membrane for the initial membrane potential equal to zero. The changes in polysialic acid flux was up to 75% after 1 ms in comparison with the zero-time flux. Both a decrease of membrane conductance and an increase of polyion chain length resulted in the diminution of this effect. An increase of praseodymium ions adsorption to positively charged liposomes and an increase of the rate of segmental movement of the -CH2 and -CH3 groups, and the choline headgrup of lipid molecules, was observed in the presence of polySia. The results show that the direction of the vectorial polyion translocation depends both on the membrane electrical properties and the degree of polymerization of the polymer, and that polysialic acid can modulate the degree of ion adsorption and the dynamics of membrane lipids.     

Translocation of Bacillus anthracis lethal and oedema factors across endosome membranes

The two exotoxins of Bacillus anthracis , the causative agent of anthrax, are the oedema toxin (PA–EF) and the lethal toxin (PA–LF). They exert their catalytic activities within the cytosol. The internalization process requires receptor-mediated endocytosis and passage through acidic vesicles. We investigated the translocation of EF and LF enzymatic moieties across the target cell membrane. By selective permeabilization of the plasma membrane with Clostridium perfringens delta-toxin, we observed free full-size lethal factor (LF) within the cytosol, resulting from specific translocation from early endosomes. In contrast, oedema factor (EF) remained associated with the membranes of vesicles.     

Translocation of positively charged copoly(Lys/Tyr) across phospholipid membranes

Much attention has recently been paid to the study of positively charged polypeptides as a possible carrier for therapeutic protein or DNA delivery to cells. In this study, we have investigated the translocation of positively charged copoly(Lys/Tyr) (MW=72000, DP=385) across lipid membranes constituted from egg-phosphatidylcholine (EPC), dioleoyl-phosphatidylethanolamine (DOPE), as well as soybean phospholipids (SBPL) using zeta potential method, circular dichroism spectroscopy (CD), electrophysiology technique, fluorescence spectroscopy, and confocal laser scanning microscopy. Results of zeta potentials show that copoly(Lys/Tyr) associate with lipid membranes and become gradually saturated on the membranes either hydrophobically or electrostatically or both. CD studies demonstrate that the copoly(Lys/Tyr) takes and remains beta-sheet conformation during its interaction with liposome membranes, indicating that the translocation process should be carpet-mode like. Data from the electrophysiology technique reveal that positively charged copoly(Lys/Tyr) can cause transmembrane currents under an applied voltage, confirming its transfer across lipid membranes. Fluorescence spectroscopy results display a three-step mechanism of translocation across membrane: adsorption, transportation, and desorption, which has been verified by results from confocal laser scanning microscopy. We provided the first direct observation that the positively charged polypeptides, copoly(Lys/Tyr), can translocate through SBPL and EPC/DOPE lipid bilayer membranes. In addition, we found that the translocation efficiency of copoly(Lys/Tyr) was higher on the EPC/DOPE lipid membrane than on the SBPL lipid membrane.     

Transmembrane transport of peptidoglycan precursors across model and bacterial membranes

Translocation of the peptidoglycan precursor Lipid II across the cytoplasmic membrane is a key step in bacterial cell wall synthesis, but hardly understood. Using NBD-labelled Lipid II, we showed by fluorescence and TLC assays that Lipid II transport does not occur spontaneously and is not induced by the presence of single spanning helical transmembrane peptides that facilitate transbilayer movement of membrane phospholipids. MurG catalysed synthesis of Lipid II from Lipid I in lipid vesicles also did not result in membrane translocation of Lipid II. These findings demonstrate that a specialized protein machinery is needed for transmembrane movement of Lipid II. In line with this, we could demonstrate Lipid II translocation in isolated Escherichia coli inner membrane vesicles and this transport could be uncoupled from the synthesis of Lipid II at low temperatures. The transport process appeared to be independent from an energy source (ATP or proton motive force). Additionally, our studies indicate that translocation of Lipid II is coupled to transglycosylation activity on the periplasmic side of the inner membrane.     

Translocation of analogues of the antimicrobial peptides magainin and buforin across human cell membranes

Cationic antimicrobial peptides play important roles in innate immunity. Compared with extensive studies on peptide-bacteria interactions, little is known about peptide-human cell interactions. Using human cervical carcinoma HeLa and fibroblastic TM12 cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47-57) peptide (YGRKKRRQRRR). The dose, time, temperature, and energy dependence of translocation suggested that the three peptides cross cell membranes through different mechanisms. The magainin peptide was internalized within a time scale of tens of minutes. The cooperative concentration dependence of uptake suggested that the peptide forms a pore as an intermediate similar to the observations in model membranes. Furthermore, the translocation was coupled with cytotoxicity, which was larger for tumor HeLa cells. In contrast, the buforin peptide translocated within 10 min by a temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the highest concentration investigated (100 microm). The uptake of the Tat peptide was proportional to the peptide concentration, and the concentration dependence was lost upon ATP depletion. The peptide exhibited a moderate cytotoxicity at higher concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa green fluorescent protein, also entered cells, suggesting a potency of the peptide as a vector for macromolecular delivery into cells. However, the mechanism appeared to be different from that of the parent peptide.     

Foreword: Translocation of proteins across membranes: systems,conservation and evolutionary origin

The interactions of mouse thymocytes with unilamellar phospholipid vesicles comprised of dimyristyl lecithin (DML), dipalmitoyl lecithin (DPL), dioleoyl lecithin (DOL), and egg yolk lecithin (EYL) were examined in vitro.

In cells treated with [³H]DML or [³H]DPL vesicles, electron microscope (EM) autoradiographic analysis showed most of the radioactive lipids to be confined to the cell surface. Transmission EM studies showed the presence of intact vesicles (DPL) and collapsed or ruptured vesicle fragments (DML) adsorbed to the surfaces of treated cells. In cells treated with DPL vesicles containing a watersoluble dye (6-carboxyfluorescein; 6-CF), most of the fluorescent vesicles were localized at the periphery of the treated cells. Furthermore, substantial fractions of the cell-associated DPL and DML could be released by a mild trypsinization without damaging the cells. These results suggest that the uptake of DML and DPL is primarily due to vesicle-cell adsorption. Such an adsorption process appears to be enhanced at or below the thermotropic-phase transition temperature of the vesicle lipid. Under certain conditions these adherent vesicles also formed patches or caps on the cell surface.

In cells treated with DOL or EYL vesicles, transmission EM and EM autoradiography showed relatively little exogenous vesicle lipid located at the cell surface. Thymocytes incubated (37°C) with [¹⁴C] EYL vesicles containing a trapped marker, [³H]inulin, incor porated both isotopes at identical rates. In separate experiments it was found that this marker was located inside the treated cells. Thymocytes treated with DOL vesicles containing 6-CF exhibited a uniform and diffuse distribution of dye in the internal volume of the cells. Little cell-associated EYL or DOL could be released by trypsinization. Evidence against endocytosis of intact vesicles as a major pathway of vesicle uptake is also presented. These observations, coupled with the demonstration of vesicle-cell lipid exchange as a minor component of vesicle uptake suggest that incorporation of EYL and DOL vesicles by thymocytes is primarily by vesicle-cell fusion.     

Protein translocation across membranes.

Cellular membranes act as semipermeable barriers to ions and macromolecules. Specialized mechanisms of transport of proteins across membranes have been developed during evolution. There are common mechanistic themes among protein translocation systems in bacteria and in eukaryotic cells. Here we review current understanding of mechanisms of protein transport across the bacterial plasma membrane as well as across several organelle membranes of yeast and mammalian cells. We consider a variety of organelles including the endoplasmic reticulum, outer and inner membranes of mitochondria, outer, inner, and thylakoid membranes of chloroplasts, peroxisomes, and lysosomes. Several common principles are evident: (a) multiple pathways of protein translocation across membranes exist, (b) molecular chaperones are required in the cytosol, inside the organelle, and often within the organelle membrane, (c) ATP and/or GTP hydrolysis is required, (d) a proton-motive force across the membrane is often required, and (e) protein translocation occurs through gated, aqueous channels. There are exceptions to each of these common principles indicating that our knowledge of how proteins translocate across membranes is not yet complete.     

Translocation of N-terminal tails across the plasma membrane.

Previously we have shown that the first hydrophobic domain of leader peptidase (lep) can function to translocate a short N-terminal 18 residue antigenic peptide from the phage Pf3 coat protein across the plasma membrane of Escherichia coli. We have now examined the mechanism of insertion of N-terminal periplasmic tails and have defined the features needed to translocate these regions. We find that short tails of up to 38 residues are efficiently translocated in a SecA- and SecY-independent manner while longer tails are very poorly inserted. Efficient translocation of a 138 residue tail is restored and is Sec-dependent by the addition of a leader sequence to the N-terminus of the protein. We also find that while there is no amphiphilic helix requirement for N-terminal translocation, there is a charge requirement that is needed within the tail; an arginine and lysine residue can inhibit or completely block translocation when introduced into the tail region. Intriguingly, the membrane potential is required for insertion of a 38 residue tail but not for a 23 residue tail.     

Translocation of bacterial proteins--an overview

Recent progress in the understanding of the nature of the extraordinary variety of protein translocation systems, mainly in Gram negative bacteria, is reviewed. This takes us from the insertion of proteins into the inner membrane via the sophisticated Sec apparatus, the lethal injection of Type III proteins into host cells and on to the beautiful machine that assembles the flagellum. Attempts are made to establish some order, some common principles that might explain the variety and the complexity of some systems. The fundamentals considered are the nature of different transport signals, the nature of translocons (a wide variety of inner membrane types, outer membrane translocons are more conserved), the process of docking to translocons, the role of chaperones and the folding of transported proteins, the energetics of translocation, and prospects for future advances.