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     

sHsps and their role in the chaperone network

Small Hsps (sHsps) encompass a widespread but diverse class of proteins. These low molecular mass proteins (15—42 kDa) form dynamic oligomeric structures ranging from 9 to 50 subunits. sHsps display chaperone function in vitro, and in addition they have been suggested to be involved in the inhibition of apoptosis, organisation of the cytoskeleton and establishing the refractive properties of the eye lens in the case of α-crystallin. How these different functions can be explained by a common mechanism is unclear at present. However, as most of the observed phenomena involve nonnative protein, the repeatedly reported chaperone properties of sHsps seem to be of key importance for understanding their function. In contrast to other chaperone families, sHsps bind several nonnative proteins per oligomeric complex, thus representing the most efficient chaperone family in terms of the quantity of substrate binding. In some cases, the release of substrate proteins from the sHsp complex is achieved in cooperation with Hsp70 in an ATP-dependent reaction, suggesting that the role of sHsps in the network of chaperones is to create a reservoir of nonnative refoldable protein.     

Control of cell growth: Rag GTPases in activation of TORC1

The target of rapamycin (TOR) is a central regulator controlling cell growth. TOR is highly conserved from yeast to mammals, and is deregulated in human cancers and diabetes. TOR complex 1 (TORC1) integrates signals from growth factors, cellular energy status, stress, and amino acids to control cell growth, mitochondrial metabolism, and lipid biosynthesis. The mechanisms of growth factors and cellular energy status in regulating TORC1 have been well established, whereas the mechanism by which amino acid induces TORC1 remains largely unknown. Recent studies revealed that Rag GTPases play a central role in the regulation of TORC1 activation in response to amino acids. In this review, we will discuss the recent progress in our understanding of Rag GTPase-regulated TORC1 activation in response to amino acids. Particular focus will be given to the function of Rag GTPases in TORC1 activation and how Rag GTPases are regulated by amino acids.     

Retinoblastoma family proteins: insights gained through genetic manipulation of mice

The retinoblastoma (Rb) gene was identified as the first tumor suppressor gene two decades ago. Since this initial discovery, it has become clear that deregulated Rb function constitutes a hallmark of human malignancies. Rb is a well-established regulator of the cell cycle. Rb has also been implicated in playing a role in a wide variety of cellular processes including DNA repair, cellular senescence, cell fate determination and apoptosis. Animals lacking Rb and/or its family members p107 and p130 have led scientists to uncover new and exciting roles for this protein family in development as well as tumor suppression. The ability to ablate Rb in a temporal and cell-type-specific manner has offered further, often unexpected, insights into Rb function. This review summarizes the phenotypic consequences of Rb family ablation in mice, and discusses how these findings contribute to the increasingly complex picture of Rb family function in development and tumor suppression. Received 11 October 2005; received after revision 16 November 2005; accepted 28 November 2005     

Direct evidence that the N-terminal extensions of the TAP complex act as autonomous interaction scaffolds for the assembly of the MHC I peptide-loading complex

The loading of antigenic peptides onto major histocompatibility complex class I (MHC I) molecules is an essential step in the adaptive immune response against virally or malignantly transformed cells. The ER-resident peptide-loading complex (PLC) consists of the transporter associated with antigen processing (TAP1 and TAP2), assembled with the auxiliary factors tapasin and MHC I. Here, we demonstrated that the N-terminal extension of each TAP subunit represents an autonomous domain, named TMD(0), which is correctly targeted to and inserted into the ER membrane. In the absence of coreTAP, each TMD(0) recruits tapasin in a 1:1 stoichiometry. Although the TMD(0)s lack known ER retention/retrieval signals, they are localized to the ER membrane even in tapasin-deficient cells. We conclude that the TMD(0)s of TAP form autonomous interaction hubs linking antigen translocation into the ER with peptide loading onto MHC I, hence ensuring a major function in the integrity of the antigen-processing machinery.     

Alpha-synuclein and Parkinson's disease

The involvement of alpha-synuclein in neurodegenerative diseases was first suspected after the isolation of an alpha-synuclein fragment (NAC) from amyloid plaques in Alzheimer's disease (AD). Later, two different alpha-synuclein mutations were shown to be associated with autosomal-dominant Parkinson's disease (PD), but only in a small number of families. However, the discovery that alpha-synuclein is a major component of Lewy bodies and Lewy neurites, the pathological hallmarks of PD, confirmed its role in PD pathogenesis. Pathological aggregation of the protein might be responsible for neurodegeneration. In addition, soluble oligomers of alpha-synuclein might be even more toxic than the insoluble fibrils found in Lewy bodies. Multiple factors have been shown to accelerate alpha-synuclein aggregation in vitro. Therapeutic strategies aimed to prevent this aggregation are therefore envisaged. Although little has been learned about its normal function, alpha-synuclein appears to interact with a variety of proteins and membrane phospholipids, and may therefore participate in a number of signaling pathways. In particular, it may play a role in regulating cell differentiation, synaptic plasticity, cell survival, and dopaminergic neurotransmission. Thus, pathological mechanisms based on disrupted normal function are also possible.     

Molecular guardians for newborn proteins: ribosome-associated chaperones and their role in protein folding

A central dogma in biology is the conversion of genetic information into active proteins. The biosynthesis of proteins by ribosomes and the subsequent folding of newly made proteins represent the last crucial steps in this process. To guarantee the correct folding of newly made proteins, a complex chaperone network is required in all cells. In concert with ongoing protein biosynthesis, ribosome-associated factors can interact directly with emerging nascent polypeptides to protect them from degradation or aggregation, to promote folding into their native structure, or to otherwise contribute to their folding program. Eukaryotic cells possess two major ribosome-associated systems, an Hsp70/Hsp40-based chaperone system and the functionally enigmatic NAC complex, whereas prokaryotes employ the Trigger Factor chaperone. Recent structural insights into Trigger Factor reveal an intricate cradle-like structure that, together with the exit site of the ribosome, forms a protected environment for the folding of newly synthesized proteins. Received 29 June 2005; received after revision 4 August 2005; accepted 18 August 2005     

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     

pHi regulatory ion transporters: an update on structure, regulation and cell function

Intracellular pH (pH_i) is a major regulator of various and critical cellular functions. A close regulation of pH_i is thus mandatory to maintain normal cellular activity. To this end, all cells express ion transporters that carry across their plasma membrane H⁺ or equivalent H⁺ into and out of the cell. Besides pH_i, these ion transporters are under the regulation of neurohormonal stimuli. This review summarises the molecular identity, regulation and function of the main membrane pH-regulatory ion transporters. Received 30 December 1998; received after revision 4 February 1999; accepted 9 February 1999     

The Arp2/3 complex: a central regulator of the actin cytoskeleton

In recent years the Arp2/3 complex has emerged as a central regulator of actin dynamics, assembling and cross-linking actin filaments to produce a diverse array of cellular structures. Here I discuss our current state of knowledge about this actin-remodelling machine. The predicted structure of the Arp2/3 complex can be directly correlated with its ability to nucleate, cap and cross-link actin filaments. A growing family of Arp2/3 complex activators such as the WASP family, type I myosins, and the newly identified activators cortactin and Abp1p tightly regulate this activity within the cell. Localised activation of the Arp2/3 complex produces structures such as lamellipodia or actin patches via a process termed dendritic nucleation. Furthermore, several pathogenic microorganisms have evolved strategies to 'hijack' the Arp2/3 complex to their own advantage. Finally, I discuss some of the questions which remain unanswered about this fascinating complex. Received 2 April 2001; received after revision 15 May 2001; accepted 18 May 2001     

Regulation of membrane trafficking and endocytosis by protein kinase C: emerging role of the pericentrion,a novel protein kinase C-dependent subset of recycling endosomes

The protein kinase C (PKC) family of isoenzymes has been shown to regulate a variety of cellular processes, including receptor desensitization and internalization, and this has sparked interest in further delineation of the roles of specific isoforms of PKC in membrane trafficking and endocytosis. Recent studies have identified a novel translocation of PKC to a juxtanuclear compartment, the pericentrion, which is distinct from the Golgi complex but epicentered on the centrosome. Sustained activation of PKC (longer than 30 min) also results in sequestration of plasma membrane lipids and proteins to the same compartment, demonstrating a global effect on endocytic trafficking. This review summarizes these studies, particularly focusing on the characterization of the pericentrion as a distinct PKC-dependent subset of recycling endosomes. We also discuss emerging insights into a role for PKC as a central hub in regulating vesicular transport pathways throughout the cell, with implications for a wide range of pathobiologic processes, e.g. diabetes and abnormal neurotransmission or receptor desensitization. Received 11 August 2006; received after revision 20 September 2006; accepted 7 November 2006     

Atomic model of human cystic fibrosis transmembrane conductance regulator: Membrane-spanning domains and coupling interfaces

We describe herein an atomic model of the outward-facing three-dimensional structure of the membrane-spanning domains (MSDs) and nucleotide-binding domains (NBDs) of human cystic fibrosis transmembrane conductance regulator (CFTR), based on the experimental structure of the bacterial transporter Sav1866. This model, which is in agreement with previous experimental data, highlights the role of some residues located in the transmembrane passages and directly involved in substrate translocation and of some residues within the intracellular loops (ICL1-ICL4) making MSD/NBD contacts. In particular, our model reveals that D173 ICL1 and N965 ICL3 likely interact with the bound nucleotide and that an intricate H-bond network (involving especially the ICL4 R1070 and the main chain of NBD1 F508) may stabilize the interface between MSD2 and the NBD1F508 region. These observations allow new insights into the ATP-binding sites asymmetry and into the molecular consequences of the F508 deletion, which is the most common cystic fibrosis mutation.     

Delta opioid receptors recycle to the membrane after sorting to the degradation path

Soon after internalization delta opioid receptors (DOPrs) are committed to the degradation path by G protein-coupled receptor (GPCR)-associated binding protein. Here we provide evidence that this classical post-endocytic itinerary may be rectified by downstream sorting decisions which allow DOPrs to regain to the membrane after having reached late endosomes (LE). The LE sorting mechanism involved ESCRT accessory protein Alix and the TIP47/Rab9 retrieval complex which supported translocation of the receptor to the TGN, from where it subsequently regained the cell membrane. Preventing DOPrs from completing this itinerary precipitated acute analgesic tolerance to the agonist DPDPE, supporting the relevance of this recycling path in maintaining the analgesic response by this receptor. Taken together, these findings reveal a post-endocytic itinerary where GPCRs that have been sorted for degradation can still recycle to the membrane.     

Structural studies of a phosphatidyl serine-amorphous calcium phosphate complex

A phosphatidyl serine-amorphous calcium phosphate complex has been synthesized as a model of the matrix vesicle system that is associated with the induction of mineral deposition in bone, cartilage and dentine. The complex has been studied using a novel technique of subtractive extended X-ray absorption fine structure (EXAFS). This enables spectra of the components of the molecules to be subtracted from the complex so as to identify the sites of interaction. The results suggest there is a movement in the nitrogen atom of the phosphatidyl serine which approaches the calcium atom in the mineral phase. This interpretation would link the membrane structure of the vesicle to the structure of the mineral in a way that could explain some of its roles in biomineralization. Received 14 November 1997; accepted 23 December 1997     

Strategies for identifying synthetic peptides to act as inhibitors of NADPH oxidases, or “All that you did and did not want to know about Nox inhibitory peptides”

Phagocytes utilize reactive oxygen species (ROS) to kill pathogenic microorganisms. The source of ROS is an enzymatic complex (the NADPH oxidase), comprising a membrane-associated heterodimer (flavocytochrome b (558)), consisting of subunits Nox2 and p22(phox), and four cytosolic components (p47(phox), p67(phox), p40(phox), and Rac). The primordial ROS (superoxide) is generated by the reduction of molecular oxygen by NADPH via redox centers located on Nox2. This process is activated by the translocation of the cytosolic components to the membrane and their assembly with Nox2. Membrane translocation is preceded by interactions among cytosolic components. A number of proteins structurally and functionally related to Nox2 have been discovered in many cells (the Nox family) and these have pleiotropic functions related to the production of ROS. An intense search is underway to design therapeutic means to modulate Nox-dependent overproduction of ROS, associated with diseases. Among drug candidates, a central position is held by synthetic peptides reflecting domains in oxidase components involved in NADPH oxidase assembly. Peptides, corresponding to domains in Nox2, p22(phox), p47(phox), and Rac, found to be oxidase activation inhibitory in vitro, are reviewed. Usually, peptides are inhibitory only when added preceding assembly of the complex. Although competition with intact components seems most likely, less obvious mechanisms are, sometimes, at work. The use of peptides as inhibitory drugs in vivo requires the development of methods to assure cell penetration, resistance to degradation, and avoidance of toxicity, and modest successes have been achieved. The greatest challenge remains the discovery of peptide inhibitors acting specifically on individual Nox isoforms.     

The sigmaS subunit of RNA polymerase as a signal integrator and network master regulator in the general stress response in Escherichia coli

The sigmaS (RpoS) subunit of RNA polymerase in Escherichia coli is a key master regulator which allows this bacterial model organism and important pathogen to adapt to and survive environmentally rough times. While hardly present in rapidly growing cells, sigmaS strongly accumulates in response to many different stress conditions, partly replaces the vegetative sigma subunit in RNA polymerase and thereby reprograms this enzyme to transcribe sigmaS-dependent genes (up to 10% of the E. coli genes). In this review, we summarize the extremely complex regulation of sigmaS itself and multiple signal input at the level of this master regulator, we describe the way in which sigmaS specifically recognizes "stress" promoters despite their similarity to vegetative promoters, and, while being far from comprehensive, we give a short overview of the far-reaching physiological impact of sigmaS. With sigmaS being a central and multiple signal integrator and master regulator of hundreds of genes organized in regulatory cascades and sub-networks or regulatory modules, this system also represents a key model system for analyzing complex cellular information processing and a starting point for understanding the complete regulatory network of an entire cell.