Protein transport across and into cell membranes in bacteria and archaea

In the three domains of life, the Sec, YidC/Oxa1, and Tat translocases play important roles in protein translocation across membranes and membrane protein insertion. While extensive studies have been performed on the endoplasmic reticular and Escherichia coli systems, far fewer studies have been done on archaea, other Gram-negative bacteria, and Gram-positive bacteria. Interestingly, work carried out to date has shown that there are differences in the protein transport systems in terms of the number of translocase components and, in some cases, the translocation mechanisms and energy sources that drive translocation. In this review, we will describe the different systems employed to translocate and insert proteins across or into the cytoplasmic membrane of archaea and bacteria.     

Biogenesis of bacterial inner-membrane proteins

All cells must traffic proteins into and across their membranes. In bacteria, several pathways have evolved to enable protein transfer across the inner membrane, the periplasm, and the outer membrane. The major route of protein translocation in and across the cytoplasmic membrane is the general secretion pathway (Sec-pathway). The biogenesis of membrane proteins not only requires protein translocation but also coordinated targeting to the membrane beforehand and folding and assembly into their protein complexes afterwards to function properly in the cell. All these processes are responsible for the biogenesis of membrane proteins that mediate essential functions of the cell such as selective transport, energy conversion, cell division, extracellular signal sensing, and motility. This review will highlight the most recent developments on the structure and function of bacterial membrane proteins, focusing on the journey that integral membrane proteins take to find their final destination in the inner membrane.     

Cell wall polymers in Archaea (Archaebacteria)

The distribution of the various cell wall and cell envelope (S-layer) polymers among the main lineages of the domain Archaea (Archaebacteria) and the chemical composition and primary structure of polymers forming rigid cell wall sacculi is described. Differences between bacteria and archaea in their sensitivity to antibiotics which inhibit cell wall synthesis in bacteria are discussed.     

The bacterial translocase: a dynamic protein channel complex

The major route of protein translocation in bacteria is the so-called general secretion pathway (Sec-pathway). This route has been extensively studied in Escherichia coli and other bacteria. The movement of preproteins across the cytoplasmic membrane is mediated by a multimeric membrane protein complex called translocase. The core of the translocase consists of a proteinaceous channel formed by an oligomeric assembly of the heterotrimeric membrane protein complex SecYEG and the peripheral adenosine triphosphatase (ATPase) SecA as molecular motor. Many secretory proteins utilize the molecular chaperone SecB for targeting and stabilization of the unfolded state prior to translocation, while most nascent inner membrane proteins are targeted to the translocase by the signal recognition particle and its membrane receptor. Translocation is driven by ATP hydrolysis and the proton motive force. In the last decade, genetic and biochemical studies have provided detailed insights into the mechanism of preprotein translocation. Recent crystallographic studies on SecA, SecB and the SecYEG complex now provide knowledge about the structural features of the translocation process. Here, we will discuss the mechanistic and structural basis of the translocation of proteins across and the integration of membrane proteins into the cytoplasmic membrane.Received 10 January 2003; received after revision 2 April 2003; accepted 4 April 2003     

Inhibition by transmembrane peptides of chimeric insulin receptors

Receptor tyrosine kinases play essential roles in cell proliferation and differentiation. We have recently shown that peptides corresponding to the transmembrane domains of the epidermal growth factor (EGF) and ErbB2 receptors inhibit their corresponding receptor activation in cancer cell lines. We extend this observation to cells transfected with chimeric insulin receptors where the transmembrane domain has been replaced by that of the EGF receptor or a mutated Erb2 domain. Peptides corresponding to the transmembrane domains of the EGF receptor and ErbB2 are able to inhibit specifically the autophosphorylation of insulin receptors with the corresponding domain. This inhibitory effect is correlated with the propensity of the different transmembrane domains to self-associate in a genetic reporter assay. Thus, our data strengthen the notion that transmembrane domains are involved in erbB receptor activation, and that these receptors can be modulated by inhibiting proteinprotein interactions within the membrane.Received 25 May 2005; received after revision 13 July 2005; accepted 22 July 2005     

New developments in the biological functions of lysophospholipids

Lysophospholipids have long been recognized as membrane phospholipid metabolites, but only recently has their role as intercellular signaling molecules been appreciated. Two of the best-studied lysophospholipids, LPA and S1P, signal through cognate G-protein-coupled receptors to activate many well-known intracellular signaling pathways, leading to a variety of biologically important cell responses. Lysophospholipids and their receptors have been found in a wide range of tissues and cell types, indicating their importance in many physiological processes, including reproduction, vascular development, cancer and nervous system function. This article will focus on the most recent findings regarding the biological functions of lysophospholipids in mammalian systems, specifically as they relate to health and disease. Received 5 April 2006; received after revision 22 June 2006; accepted 9 August 2006     

Colicins: prokaryotic killer-pores.

Colicins are plasmid-encoded protein antibiotics which kill bacteria closely related to the producing strain (generally Escherichia coli). The study of the function of colicins has revealed many features which reflect common targeting and translocation mechanisms with bacteriophages and toxins. Like many toxins, colicins are composed of structural domains specialized in one of the different steps of the activity, targeting, translocation and killing. The major group comprises those colicins which permeabilize the cytoplasmic membrane, thereby destroying the cell's membrane potential. These colicins form well-defined voltage-gated ion channels in artificial membranes. The scope of this review is to describe some of the more recent findings concerning the structure and mode of action of pore-forming colicins with a special attention to models of membrane insertion and pore structure based on the recently determined three-dimensional structure of the pore-forming domain of colicin A.     

The transverse-axial tubular system of cardiomyocytes

A characteristic histological feature of striated muscle cells is the presence of deep invaginations of the plasma membrane (sarcolemma), most commonly referred to as T-tubules or the transverse-axial tubular system (TATS). TATS mediates the rapid spread of the electrical signal (action potential) to the cell core triggering Ca²⁺ release from the sarcoplasmic reticulum, ultimately inducing myofilament contraction (excitation–contraction coupling). T-tubules, first described in vertebrate skeletal muscle cells, have also been recognized for a long time in mammalian cardiac ventricular myocytes, with a structure and a function that in recent years have been shown to be far more complex and pivotal for cardiac function than initially thought. Renewed interest in T-tubule function stems from the loss and disorganization of T-tubules found in a number of pathological conditions including human heart failure (HF) and dilated and hypertrophic cardiomyopathies, as well as in animal models of HF, chronic ischemia and atrial fibrillation. Disease-related remodeling of the TATS leads to asynchronous and inhomogeneous Ca²⁺-release, due to the presence of orphan ryanodine receptors that have lost their coupling with the dihydropyridine receptors and are either not activated or activated with a delay. Here, we review the physiology of the TATS, focusing first on the relationship between function and structure, and then describing T-tubular remodeling and its reversal in disease settings and following effective therapeutic approaches.     

Fluorescence spectroscopy and molecular dynamics simulations in studies on the mechanism of membrane destabilization by antimicrobial peptides

Since their initial discovery, 30 years ago, antimicrobial peptides (AMPs) have been intensely investigated as a possible solution to the increasing problem of drug-resistant bacteria. The interaction of antimicrobial peptides with the cellular membrane of bacteria is the key step of their mechanism of action. Fluorescence spectroscopy can provide several structural details on peptide–membrane systems, such as partition free energy, aggregation state, peptide position and orientation in the bilayer, and the effects of the peptides on the membrane order. However, these “low-resolution” structural data are hardly sufficient to define the structural requirements for the pore formation process. Molecular dynamics simulations, on the other hand, provide atomic-level information on the structure and dynamics of the peptide–membrane system, but they need to be validated experimentally. In this review we summarize the information that can be obtained by both approaches, highlighting their versatility and complementarity, suggesting that their synergistic application could lead to a new level of insight into the mechanism of membrane destabilization by AMPs.     

Signal perception in plant pathogen defense

Highly sensitive and specific recognition systems for microbial pathogens are essential for disease resistance in plants. Structurally diverse elicitors from various pathogens have been identified and shown to trigger plant defense mechanisms. Elicitor recognition by the plant is assumed to be mediated by receptors. Plant receptors for fungus-derived elicitors appear to reside preferentially in the plasma membrane, whereas viral and bacterial elicitors may enter the plant cell and are perceived intracellularly. Receptor activation initiates an intracellular signal transduction cascade leading to stimulation of a characteristic set of plant defense responses. Isolation of plant elicitor receptors and their encoding genes is expected to provide significant information on the molecular basis of signal perception and intracellular signal generation in plant-pathogen interactions.     

Hormone-induced subcellular redistribution of trimeric G proteins

Trimeric guanine nucleotide-binding proteins (G proteins) function as the key regulatory elements in a number of transmembrane signaling cascades where they convey information from agonist-activated receptors to effector molecules. The subcellular localization of G proteins is directly related to their functional role, i.e., the dominant portion of the cellular pool of G proteins resides in the plasma membrane. An intimate association of G protein subunits with the plasma membrane has been well known for a long time. However, results of a number of independent studies published in the past decade have indicated clearly that exposure of intact target cells to agonists results in subcellular redistribution of the cognate G proteins from plasma membranes to the light-vesicular membrane fractions, in internalization from the cell surface into the cell interior and in transfer from the membrane to the soluble cell fraction (high-speed supernatant), i.e., solubilization. Solubilization of G protein α subunits as a consequence of stimulation of G protein-coupled receptors (GPCRs) with agonists has also been observed in isolated membrane preparations. The membrane-cytosol shift of G proteins was detected even after direct activation of these proteins by non-hydrolyzable analogues of GTP or by cholera toxin-induced ADP-ribosylation. In addition, prolonged stimulation of GPCRs with agonists has been shown to lead to down-regulation of the relevant G proteins. Together, these data suggest that G proteins might potentially participate in a highly complex set of events, which are generally termed desensitization of the hormone response. Internalization, subcellular redistribution, solubilization, and down-regulation of trimeric G proteins may thus provide an additional means (i.e., beside receptor-based mechanisms) to dampen the hormone or neurotransmitter response after sustained (long-term) exposure. Received 31 August 2001; received after revision 31 October 2001; accepted 7 November 2001     

Processing of heparanase is mediated by syndecan-1 cytoplasmic domain and involves syntenin and α-actinin

Heparanase activity plays a decisive role in cell dissemination associated with cancer metastasis. Cellular uptake of heparanase is considered a pre-requisite for the delivery of latent 65-kDa heparanase to lysosomes and its subsequent proteolytic processing and activation into 8- and 50-kDa protein subunits by cathepsin L. Heparan sulfate proteoglycans, and particularly syndecan, are instrumental for heparanase uptake and activation, through a process that has been shown to occur independent of rafts. Nevertheless, the molecular mechanism underlying syndecan-mediated internalization outside of rafts is unclear. Here, we examined the role of syndecan-1 cytoplasmic domain in heparanase processing, utilizing deletion constructs lacking the entire cytoplasmic domain (Delta), the conserved (C1 or C2), or variable (V) regions. Heparanase processing was markedly increased following syndecan-1 over-expression; in contrast, heparanase was retained at the cell membrane and its processing was impaired in cells over-expressing syndecan-1 deleted for the entire cytoplasmic tail. We have next revealed that conserved domain 2 (C2) and variable (V) regions of syndecan-1 cytoplasmic tail mediate heparanase processing. Furthermore, we found that syntenin, known to interact with syndecan C2 domain, and α actinin are essential for heparanase processing.     

Olfactory receptor trafficking to the plasma membrane

Olfactory receptors typically exhibit poor plasma membrane localization and functionality when heterologously expressed in most cell types. It has therefore proven difficult to effectively study olfactory receptor pharmacology and signaling mechanisms using traditional cell culture systems. Over the past few years, a variety of distinct proteins have been reported to interact with olfactory receptors and facilitate olfactory receptor trafficking to the plasma membrane in heterologous cells. Advances in this area have shed significant light on the fundamental factors governing the cell-specific control of olfactory receptor trafficking.     

B7-H6/NKp30 interaction: a mechanism of alerting NK cells against tumors

Natural killer (NK) cells are lymphocytes of the innate immune system that sense target cells through a panel of activating and inhibitory receptors. Together with NKG2D, the natural cytotoxicity receptors (NCRs) are major activating receptors involved in tumor cell detection. Although numerous NKG2D ligands have been identified, characterization of the molecules interacting with the NCRs is still incomplete. The identification of B7-H6 as a counter structure of the NCR NKp30 shed light on the molecular basis of NK cell immunosurveillance. We review here the current knowledge on NKp30 and B7-H6, and we discuss their potential role in anti-tumor immunity.     

Friedrich Miescher Prize awardee lecture review. A conserved family of nuclear export receptors mediates the exit of messenger RNA to the cytoplasm

The distinguishing feature of eukaryotic cells is the segregation of RNA biogenesis and DNA replication in the nucleus, separate from the cytoplasmic machinery for protein synthesis. As a consequence, messenger RNAs (mRNAs) and all cytoplasmic RNAs from nuclear origin need to be transported from their site of synthesis in the nucleus to their final cytoplasmic destination. Nuclear export occurs through nuclear pore complexes (NPCs) and is mediated by saturable transport receptors, which shuttle between the nucleus and cytoplasm. The past years have seen great progress in the characterization of the mRNA export pathway and the identification of proteins involved in this process. A novel family of nuclear export receptors (the NXF family), distinct from the well-characterized family of importin β-like proteins, has been implicated in the export of mRNA to the cytoplasm. Received 23 January 2001; received after revision 12 April 2001; accepted 12 April 2001     

Tumor suppressor role of protein 4.1B/DAL-1

Protein 4.1B/DAL-1 is a membrane skeletal protein that belongs to the protein 4.1 family. Protein 4.1B/DAL-1 is localized to sites of cell–cell contact and functions as an adapter protein, linking the plasma membrane to the cytoskeleton or associated cytoplasmic signaling effectors and facilitating their activities in various pathways. Protein 4.1B/DAL-1 is involved in various cytoskeleton-associated processes, such as cell motility and adhesion. Moreover, protein 4.1B/DAL-1 also plays a regulatory role in cell growth, differentiation, and the establishment of epithelial-like cell structures. Protein 4.1B/DAL-1 is normally expressed in multiple human tissues, but loss of its expression or prominent down-regulation of its expression is frequently observed in corresponding tumor tissues and tumor cell lines, suggesting that protein 4.1B/DAL-1 is involved in the molecular pathogenesis of these tumors and acts as a potential tumor suppressor. This review will focus on the structure of protein 4.1B/DAL-1, 4.1B/DAL-1-interacting molecules, 4.1B/DAL-1 inactivation and tumor progression, and anti-tumor activity of the 4.1B/DAL-1.     

Extended and bent conformations of the mannose receptor family

In mammals, the mannose receptor family consists of four members, Endo180, DEC-205, phospholipase A₂ receptor and the mannose receptor. The extracellular domains of all these receptors contain a similar arrangement of domains in which an Nterminal cysteine-rich domain is followed by a single fibronectin type II domain and eight or ten C-type lectin-like domains. This review focuses on the threedimensional structure of the receptors in the mannose receptor family and its functional implication. Recent research has revealed that several members of this family can exist in at least two configurations: an extended conformation with the N-terminal cysteinerich domain pointing outwards from the cell membrane and a bent conformation where the N-terminal domains fold back to interact with C-type lectin-like domains at the middle of the structure. Conformational transitions between these two states seem to regulate the interaction of these receptors with ligands and their oligomerization. Received 25 October 2007; received after revision 23 November 2007; accepted 7 December 2007     

The expansion of GPCR transactivation-dependent signalling to include serine/threonine kinase receptors represents a new cell signalling frontier

G protein-coupled receptor (GPCR) signalling is mediated through transactivation-independent signalling pathways or the transactivation of protein tyrosine kinase receptors and the recently reported activation of the serine/threonine kinase receptors, most notably the transforming growth factor-β receptor family. Since the original observation of GPCR transactivation of protein tyrosine kinase receptors, there has been considerable work on the mechanism of transactivation and several pathways are prominent. These pathways include the “triple membrane bypass” pathway and the generation of reactive oxygen species. The recent recognition of GPCR transactivation of serine/threonine kinase receptors enormously broadens the GPCR signalling paradigm. It may be predicted that the transactivation of serine/threonine kinase receptors would have mechanistic similarities with transactivation of tyrosine kinase pathways; however, initial studies suggest that these two transactivation pathways are mechanistically distinct. Important questions are the relative importance of tyrosine and serine/threonine transactivation pathways, the contribution of transactivation to overall GPCR signalling, mechanisms of transactivation and the range of cell types in which this phenomenon occurs. The ultimate significance of transactivation-dependent signalling remains to be defined but it appears to be prominent and if so will represent a new cell signalling frontier.     

Olfactory ensheathing cells are attracted to, and can endocytose, bacteria

Olfactory ensheathing cells (OECs) have been shown previously to express Toll-like receptors and to respond to bacteria by translocating nuclear factor-kappaB from the cytoplasm to the nucleus. In this study, we show that OECs extended significantly more pseudopodia when they were exposed to Escherichia coli than in the absence of bacteria (p=0.019). Co-immunoprecipitation showed that E. coli binding to OECs was mediated by Toll-like receptor 4. Lyso-Tracker, a fluorescent probe that accumulates selectively in lysosomes, and staining for type 1 lysosome-associated membrane proteins demonstrated that endocytosed FITC-conjugated E. coli were translocated to lysosomes. They appeared to be subsequently broken down, as shown by transmission electron microscopy. No obvious adherence to the membrane and less phagocytosis was observed when OECs were incubated with inert fluorescent microspheres. The ability of OECs to endocytose bacteria supports the notion that OECs play an innate immune function by protecting olfactory tissues from bacterial infection.