Flexibility in targeting and insertion during bacterial membrane protein biogenesis

The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E. coli IMPs known to follow distinct targeting and insertion pathways. The modified model IMPs were analyzed for altered routing using an in vivo protease mapping approach. The data suggest a facultative use of different combinations of factors.     

A tale of two GTPases in cotranslational protein targeting

Guanosine triphosphatases (GTPases) comprise a superfamily of proteins that provide molecular switches to regulate numerous cellular processes. The "GTPase switch" paradigm, in which a GTPase acts as a bimodal switch that is turned "on" and "off" by external regulatory factors, has been used to interpret the regulatory mechanism of many GTPases. Recent work on a pair of GTPases in the signal recognition particle (SRP) pathway has revealed a distinct mode of GTPase regulation. Instead of the classical GTPase switch, the two GTPases in the SRP and SRP receptor undergo a series of conformational changes during their dimerization and reciprocal activation. Each conformational rearrangement provides a point at which these GTPases can communicate with and respond to their upstream and downstream biological cues, thus ensuring the spatial and temporal precision of all the molecular events in the SRP pathway. We suggest that the SRP and SRP receptor represent an emerging class of "multistate" regulatory GTPases uniquely suited to provide exquisite control over complex cellular pathways that require multiple molecular events to occur in a highly coordinated fashion.     

Molecular mechanism of co-translational protein targeting by the signal recognition particle

The signal recognition particle (SRP) is a key component of the cellular machinery that couples the ongoing synthesis of proteins to their proper localization, and has often served as a paradigm for understanding the molecular basis of protein localization within the cell. The SRP pathway exemplifies several key molecular events required for protein targeting to cellular membranes: the specific recognition of signal sequences on cargo proteins, the efficient delivery of cargo to the target membrane, the productive unloading of cargo to the translocation machinery and the precise spatial and temporal coordination of these molecular events. Here we highlight recent advances in our understanding of the molecular mechanisms underlying this pathway, and discuss new questions raised by these findings.     

mRNA targeting eliminates the need for the signal recognition particle during membrane protein insertion in bacteria

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Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane

Nascent polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytosolic, integral membrane or exported proteins. Extensive genetic and biochemical analyses have significantly expanded our knowledge of chaperone tasking throughout this process. In bacteria, it is known that the folding of newly-synthesized cytosolic proteins is mainly orchestrated by three highly conserved molecular chaperones, namely Trigger Factor (TF), DnaK (HSP70) and GroEL (HSP60). Yet, it has been reported that these major chaperones are strongly involved in protein translocation pathways as well. This review describes such essential molecular chaperone functions, with emphasis on both the biogenesis of inner membrane proteins and the post-translational targeting of presecretory proteins to the Sec and the twin-arginine translocation (Tat) pathways. Critical interplay between TF, DnaK, GroEL and other molecular chaperones and targeting factors, including SecB, SecA, the signal recognition particle (SRP) and the redox enzyme maturation proteins (REMPs) is also discussed. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

信号识别颗粒(SRP)在膜蛋白定位中的作用

依赖信号识别颗粒(signal recognition particle,SRP)的共翻译转运是所有生命体中的一个保守途径,它将新生肽链的翻译与转运耦联在一起。超过30%的新合成的多肽链被SRP转运到正确位置。最近的研究表明,大肠杆菌中SRP抑制子可以规避SRP的需求。当SRP缺失时,翻译控制在介导膜蛋白定位方面起着关键作用。本综述总结了SRP底物如何在存在或缺失SRP的情况下转运到适当的位置以及翻译速率降低如何补偿SRP的缺失。我们还讨论了不同蛋白质对SRP的依赖程度。这一回顾将为进一步研究SRP功能及膜蛋白定位提供新思路。     

Sec-dependent protein translocation across biological membranes: evolutionary conservation of an essential protein transport pathway (Review)

All living organisms, no matter how simple or complex, possess the ability to translocate proteins across biological membranes and into different cellular compartments. Although a range of membrane transport processes exist, the major pathway used to translocate proteins across the bacterial cytoplasmic membrane or the eukaryotic endoplasmic reticulum membrane is conserved and is known as the Sec or Sec61 pathway, respectively. Over the past two decades the Sec and Sec61 pathways have been studied extensively and are well characterised at the genetic and biochemical levels. However, it is only now with the recent structural determination of a number of the key elements of the pathways that the translocation complex is beginning to give up its secrets in exquisite molecular detail. This article will focus on the routes of Sec- and Sec61-dependent membrane targeting and the nature of the translocation channel in bacteria and eukaryotes.     

Synergistic actions between the SRP RNA and translating ribosome allow efficient delivery of the correct cargos during cotranslational protein targeting

During cotranslational protein targeting by the Signal Recognition Particle (SRP), the correct cargo accelerates stable complex assembly between the SRP and SRP receptor (FtsY) by several orders of magnitude, thus enabling rapid and faithful cargo delivery to the target membrane. The molecular mechanism underlying this cargo-induced rate acceleration has been unclear. Here we show that the SRP RNA allows assembly of the SRP-FtsY complex to be specifically stimulated by a correct cargo, and, reciprocally, a correct cargo enables the SRP RNA to optimize its electrostatic interactions with FtsY. These results combined with recent structural work led us to suggest a "conformational selection" model that explains the synergistic action of the SRP RNA with the cargo in accelerating complex assembly. In addition to its previously proposed role in preventing the premature dissociation of SRP and FtsY, we found that the SRP RNA also plays an active role in ensuring the formation of productive assembly intermediates, thus guiding the SRP and FtsY through the most efficient pathway of assembly.     

Oligomeric states of the SecA and SecYEG core components of the bacterial Sec translocon

Many proteins synthesized in the cytoplasm ultimately function in non-cytoplasmic locations. In Escherichia coli, the general secretory (Sec) pathway transports the vast majority of these proteins. Two fundamental components of the Sec transport pathway are the SecYEG heterotrimeric complex that forms the channel through the cytoplasmic membrane, and SecA, the ATPase that drives the preprotein to and across the membrane. This review focuses on what is known about the oligomeric states of these core Sec components and how the oligomeric state might change during the course of the translocation of a preprotein.     

Protein targeting to the bacterial cytoplasmic membrane.

Proteins that perform their activity within the cytoplasmic membrane or outside this cell boundary must be targeted to the translocation site prior to their insertion and/or translocation. In bacteria, several targeting routes are known; the SecB- and the signal recognition particle-dependent pathways are the best characterized. Recently, evidence for the existence of a third major route, the twin-Arg pathway, was gathered. Proteins that use either one of these three different pathways possess special features that enable their specific interaction with the components of the targeting routes. Such targeting information is often contained in an N-terminal extension, the signal sequence, but can also be found within the mature domain of the targeted protein. Once the nascent chain starts to emerge from the ribosome, competition for the protein between different targeting factors begins. After recognition and binding, the targeting factor delivers the protein to the translocation sites at the cytoplasmic membrane. Only by means of a specific interaction between the targeting component and its receptor is the cargo released for further processing and translocation. This mechanism ensures the high-fidelity targeting of premembrane and membrane proteins to the translocation site.     

SecG is an auxiliary component of the protein export apparatus of Escherichia coli

SecY, SecE and SecG form the membrane-embedded core complex of the Escherichia coli protein export apparatus. These three proteins co-purify and can be co-immunoprecipitated, demonstrating that they are closely associated. While SecE and SecY are generally accepted as essential components of translocase, the role of SecG is more ambiguous. It is commonly believed that deletion of secG causes a cold-sensitive phenotype and a severe defect in export, even though some reports have indicated otherwise. However, we demonstrate that deletion of secG does not produce a cold-sensitive phenotype or a strong export defect in most genetic backgrounds. The more common result is that deletion of secG causes only a mild export defect and does not result in conditional lethality. We propose that the role of SecG is not fundamental to the export process, but is merely auxiliary – as suggested previously by biochemical data – and is physiologically important only when cells are otherwise compromised. Received: 22 July 1999 / Accepted: 11 November 1999     

Cryo-electron Microscopic Structure of SecA Protein Bound to the 70S Ribosome

SecA is an ATP-dependent molecular motor pumping secretory and outer membrane proteins across the cytoplasmic membrane in bacteria. SecA associates with the protein-conducting channel, the heterotrimeric SecYEG complex, in a so-called posttranslational manner. A recent study further showed binding of a monomeric state of SecA to the ribosome. However, the true oligomeric state of SecA remains controversial because SecA can also form functional dimers, and high-resolution crystal structures exist for both the monomer and the dimer. Here we present the cryo-electron microscopy structures of Escherichia coli SecA bound to the ribosome. We show that not only a monomeric SecA binds to the ribosome but also that two copies of SecA can be observed that form an elongated dimer. Two copies of SecA completely surround the tunnel exit, providing a unique environment to the nascent polypeptides emerging from the ribosome. We identified the N-terminal helix of SecA required for a stable association with the ribosome. The structures indicate a possible function of the dimeric form of SecA at the ribosome.     

Interaction surface and topology of Get3-Get4-Get5 protein complex, involved in targeting tail-anchored proteins to endoplasmic reticulum

Recent work has uncovered the "GET system," which is responsible for endoplasmic reticulum targeting of tail-anchored proteins. Although structural information and the individual roles of most components of this system have been defined, the interactions and interplay between them remain to be elucidated. Here, we investigated the interactions between Get3 and the Get4-Get5 complex from Saccharomyces cerevisiae. We show that Get3 interacts with Get4-Get5 via an interface dominated by electrostatic forces. Using isothermal titration calorimetry and small-angle x-ray scattering, we further demonstrate that the Get3 homodimer interacts with two copies of the Get4-Get5 complex to form an extended conformation in solution.     

Complex protein targeting to dinoflagellate plastids

Protein trafficking pathways to plastids are directed by N-terminal targeting peptides. In plants this consists of a relatively simple transit peptide, while in organisms with secondary plastids (which reside within the endomembrane system) a signal peptide is appended to the transit peptide. Despite amino acid compositional differences between organisms, often due to nucleotide biases, the features of plastid targeting sequences are generally consistent within species. Dinoflagellate algae deviate from this trend. We have conducted an expressed sequence tag (EST) survey of the peridinin-plastid containing dinoflagellate Heterocapsa triquetra to identify and characterize numerous targeting presequences of plastid proteins encoded in the nucleus. Consistent with targeting systems present in other secondary plastid-containing organisms, these all possess a canonical signal peptide at their N termini, however two major classes of transit peptides occur. Both classes possess a common N-terminal portion of the transit peptide, but one class of transit peptides contains a hydrophobic domain that has been reported to act as a stop-transfer membrane anchor, temporarily arresting protein insertion into the endoplasmic reticulum. A second class of transit peptide lacks this feature. These two classes are represented approximately equally, and for any given protein the class is conserved across all dinoflagellate taxa surveyed to date. This dichotomy suggests that two mechanisms, perhaps even trafficking routes, may direct proteins to dinoflagellate plastids. A four-residue phenylalanine-based motif is also a consistent feature of H. triquetra transit peptides, which is an ancient feature predating red algae and galucophytes that was lost in green plastids.     

Signals involved in targeting membrane proteins to synaptic vesicles

1. Synaptic vesicles (SVs) mediate fast regulated secretion of classical neurotransmitters. In order to perform their task SVs rely on a restrict set of membrane proteins. The mechanisms responsible for targeting these proteins to the SV membrane are still poorly understood.2. Likewise, little is known about the intracellular routes taken by these proteins in their way to SV membrane. Recently, several domains and motifs necessary for correct localization of SV proteins have been identified.3. In this review we summarize the sequence motifs that have been identified in the cytoplasmic domains of SV proteins that are involved in endocytosis and targeting of SVs. We suggest that the vesicular acetylcholine transporter, a protein found predominantly in synaptic vesicles, is perhaps a model protein to understand the pathways and interactions that are used for synaptic vesicle targeting.     

Competitive binding of the SecA ATPase and ribosomes to the SecYEG translocon

During co-translational membrane insertion of membrane proteins with large periplasmic domains, the bacterial SecYEG complex needs to interact both with the ribosome and the SecA ATPase. Although the binding sites for SecA and the ribosome overlap, it has been suggested that these ligands can interact simultaneously with SecYEG. We used surface plasmon resonance and fluorescence correlation spectroscopy to examine the interaction of SecA and ribosomes with the SecYEG complex present in membrane vesicles and the purified SecYEG complex present in a detergent-solubilized state or reconstituted into nanodiscs. Ribosome binding to the SecYEG complex is strongly stimulated when the ribosomes are charged with nascent chains of the monotopic membrane protein FtsQ. This binding is competed by an excess of SecA, indicating that binding of SecA and ribosomes to SecYEG is mutually exclusive.     

Tools for the study of ribosome-borne protein folding activity

In addition to its role in protein synthesis, which involves a peptidyl transferase activity, the ribosome has also been described to be able to assist protein folding, at least in vitro, as presented in a Research Highlight (Das, et al., Biotechnol. J. 2008). This in vitro-described ribosome-borne protein folding activity (RPFA) is yet poorly characterized in vivo, in part because of the lack of tools to study its biological significance. There is substantial evidence documenting RPFA in vitro, and an assay intended to detect this activity in vivo has been set up in bacteria, but this assay is indirect. In this review, we describe the different tools and tests currently available to study RPFA. We put a special emphasis on the various available inhibitors of this activity and in particular, we discuss the use of 6-aminophenanthridine (6AP) and guanabenz (GA), two antiprion drugs that were very recently shown to specifically inhibit RPFA in vitro without any significant effect on the activity of the ribosome in protein synthesis. Therefore, these drugs should allow determining the potential biological role of RPFA. Importantly, the biological activity of 6AP and GA suggest a possible involvement of RPFA in human proteinopathies.     

Targeting of the human adrenoleukodystrophy protein to the peroxisomal membrane by an internal region containing a highly conserved motif

In this study we addressed the targeting requirements of peroxisomal ABC transporters, in particular the human adrenoleukodystrophy protein. This membrane protein is defective or missing in X-linked adrenoleukodystrophy, a neurodegenerative disorder predominantly presenting in childhood. Using adrenoleukodystrophy protein deletion constructs and green fluorescent protein fusion constructs we identified the amino acid regions 1-110 and 67-164 to be sufficient for peroxisomal targeting. However, the minimal region shared by these constructs (amino acids 67-110) is not sufficient for peroxisomal targeting by itself. Additionally, the NH2-terminal 66 amino acids enhance targeting efficiency. Green fluorescent protein-labeled fragments of human peroxisomal membrane protein 69 and Saccharomyces cerevisiae Pxa1 corresponding to the amino acid 67-164 adrenoleukodystrophy protein region were also directed to the mammalian peroxisome. The required region contains a 14-amino-acid motif (71-84) conserved between the adrenoleukodystrophy protein and human peroxisomal membrane protein 69 and yeast Pxa1. Omission or truncation of this motif in the adrenoleukodystrophy protein abolished peroxisomal targeting. The single amino acid substitution L78F resulted in a significant reduction of targeting efficiency. The in-frame deletion of three amino acids (del78-80LLR) within the proposed targeting motif in two patients suffering from X-linked adrenoleukodystrophy resulted in the mislocalization of a green fluorescent protein fusion protein to nucleus, cytosol and mitochondria. Our data define the targeting region of human adrenoleukodystrophy protein containing a highly conserved 14-amino-acid motif.     

Unraveling the components of protein translocation pathway in human malaria parasite Plasmodium falciparum

The targeting and translocation of proteins is an essentially required and conserved process in all the living organisms. This complex process involves multiple steps and requires a variety of factors before the protein reaches its final destination. The major components of translocation machinery are signal recognition particle (SRP) and secretory (Sec) complex. These are composed of highly conserved components. SRP contains SRP RNA and other polypeptides such as SRP9, SRP14, SRP19 and SRP54. Sec complex is composed of Sec61αβγ, Sec62 and Sec63. In this review using bioinformatics approach we have shown that the P. falciparum genome contains the homologues for all of these and other factors such as SRP receptor, and TRAM (translocation associated membrane protein), which are required for post- and co-translational protein translocation. We have also shown the various steps of translocation in a hypothetical model.