Co-translational protein targeting to the bacterial membrane

Co-translational protein targeting by the Signal Recognition Particle (SRP) is an essential cellular pathway that couples the synthesis of nascent proteins to their proper cellular localization. The bacterial SRP, which contains the minimal ribonucleoprotein core of this universally conserved targeting machine, has served as a paradigm for understanding the molecular basis of protein localization in all cells. In this review, we highlight recent biochemical and structural insights into the molecular mechanisms by which fundamental challenges faced by protein targeting machineries are met in the SRP pathway. Collectively, these studies elucidate how an essential SRP RNA and two regulatory GTPases in the SRP and SRP receptor (SR) enable this targeting machinery to recognize, sense and respond to its biological effectors, i.e. the cargo protein, the target membrane and the translocation machinery, thus driving efficient and faithful co-translational protein targeting. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

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.     

Regulation of cargo recognition,commitment, and unloading drives cotranslational protein targeting

Efficient and accurate protein localization is essential to cells and requires protein-targeting machineries to both effectively capture the cargo in the cytosol and productively unload the cargo at the membrane. To understand how these challenges are met, we followed the interaction of translating ribosomes during their targeting by the signal recognition particle (SRP) using a site-specific fluorescent probe in the nascent protein. We show that initial recruitment of SRP receptor (SR) selectively enhances the affinity of SRP for correct cargos, thus committing SRP-dependent substrates to the pathway. Real-time measurement of cargo transfer from the targeting to translocation machinery revealed multiple factors that drive this event, including GTPase rearrangement in the SRP–SR complex, stepwise displacement of SRP from the ribosome and signal sequence by SecYEG, and elongation of the nascent polypeptide. Our results elucidate how active and sequential regulation of the SRP–cargo interaction drives efficient and faithful protein targeting.     

Signal recognition particle mediated protein targeting in Escherichia coli

The signal recognition particle (SRP) is a conserved ribonucleoprotein complex that binds to targeting sequences in nascent secretory and membrane proteins. The SRP guides these proteins to the cytoplasmic membrane in prokaryotes and the endoplasmic reticulum membrane in eukaryotes via an interaction with its cognate receptor. The E. coli SRP is relatively small and is currently used as a model for fundamental and applied studies on translation-linked protein targeting. In this review recent advances in our understanding of the structure and function of the E. coli SRP and its receptor are discussed. In particular, the interplay between the SRP pathway and other targeting routes, the role of guanine nucleotides in cycling of the SRP and the substrate specificity of the SRP are highlighted     

Translation elongation regulates substrate selection by the signal recognition particle

The signal recognition particle (SRP) is a universally conserved cellular machinery responsible for delivering membrane and secretory proteins to the proper cellular destination. The precise mechanism by which fidelity is achieved by the SRP pathway within the in vivo environment is yet to be understood. Previous studies have focused on the SRP pathway in isolation. Here we describe another important factor that modulates substrate selection by the SRP pathway: the ongoing synthesis of the nascent polypeptide chain by the ribosome. A slower translation elongation rate rescues the targeting defect of substrate proteins bearing mutant, suboptimal signal sequences both in vitro and in vivo. Consistent with a kinetic origin of this effect, similar rescue of protein targeting was also observed with mutant SRP receptors or SRP RNAs that specifically compromise the kinetics of SRP-receptor interaction during protein targeting. These data are consistent with a model in which ongoing protein translation is in constant kinetic competition with the targeting of the nascent proteins by the SRP and provides an important factor to regulate the fidelity of substrate selection by the SRP.     

Lipid activation of the signal recognition particle receptor provides spatial coordination of protein targeting

The signal recognition particle (SRP) and SRP receptor comprise the major cellular machinery that mediates the cotranslational targeting of proteins to cellular membranes. It remains unclear how the delivery of cargos to the target membrane is spatially coordinated. We show here that phospholipid binding drives important conformational rearrangements that activate the bacterial SRP receptor FtsY and the SRP–FtsY complex. This leads to accelerated SRP–FtsY complex assembly, and allows the SRP–FtsY complex to more efficiently unload cargo proteins. Likewise, formation of an active SRP–FtsY GTPase complex exposes FtsY’s lipid-binding helix and enables stable membrane association of the targeting complex. Thus, membrane binding, complex assembly with SRP, and cargo unloading are inextricably linked to each other via conformational changes in FtsY. These allosteric communications allow the membrane delivery of cargo proteins to be efficiently coupled to their subsequent unloading and translocation, thus providing spatial coordination during protein targeting.     

Vestiges of the Bacterial Signal Recognition Particle-Based Protein Targeting in Mitochondria

The main bacterial pathway for inserting proteins into the plasma membrane relies on the signal recognition particle (SRP), composed of the Ffh protein and an associated RNA component, and the SRP-docking protein FtsY. Eukaryotes use an equivalent system of archaeal origin to deliver proteins into the endoplasmic reticulum, whereas a bacteria-derived SRP and FtsY function in the plastid. Here we report on the presence of homologs of the bacterial Ffh and FtsY proteins in various unrelated plastid-lacking unicellular eukaryotes, namely Heterolobosea, Alveida, Goniomonas, and Hemimastigophora. The monophyly of novel eukaryotic Ffh and FtsY groups, predicted mitochondrial localization experimentally confirmed for Naegleria gruberi, and a strong alphaproteobacterial affinity of the Ffh group, collectively suggest that they constitute parts of an ancestral mitochondrial signal peptide-based protein-targeting system inherited from the last eukaryotic common ancestor, but lost from the majority of extant eukaryotes. The ability of putative signal peptides, predicted in a subset of mitochondrial-encoded N. gruberi proteins, to target a reporter fluorescent protein into the endoplasmic reticulum of Trypanosoma brucei, likely through their interaction with the cytosolic SRP, provided further support for this notion. We also illustrate that known mitochondrial ribosome-interacting proteins implicated in membrane protein targeting in opisthokonts (Mba1, Mdm38, and Mrx15) are broadly conserved in eukaryotes and nonredundant with the mitochondrial SRP system. Finally, we identified a novel mitochondrial protein (MAP67) present in diverse eukaryotes and related to the signal peptide-binding domain of Ffh, which may well be a hitherto unrecognized component of the mitochondrial membrane protein-targeting machinery.     

Novel proteomic tools reveal essential roles of SRP and importance of proper membrane protein biogenesis

The signal recognition particle (SRP), which mediates cotranslational protein targeting to cellular membranes, is universally conserved and essential for bacterial and mammalian cells. However, the current understanding of the role of SRP in cell physiology and pathology is still poor, and the reasons behind its essential role in cell survival remain unclear. Here, we systematically analyzed the consequences of SRP loss in E. coli using time-resolved quantitative proteomic analyses. A series of snapshots of the steady-state and newly synthesized proteome unveiled three stages of cellular responses to SRP depletion, and demonstrated essential roles of SRP in metabolism, membrane potential, and protein and energy homeostasis in both the membrane and cytoplasm. We also identified a group of periplasmic proteins, including key molecular chaperones, whose localization was impaired by the loss of SRP; this and additional results showed that SRP is crucial for protein homeostasis in the bacterial envelope. These results reveal the extensive roles that SRP plays in bacterial physiology, emphasize the importance of proper membrane protein biogenesis, and demonstrate the ability of time-resolved quantitative proteomic analysis to provide new biological insights.     

Dissecting the roles of Cse1 and Nup2 in classical NLS‐cargo release in vivo

The importin α/β transport machinery mediates the nuclear import of cargo proteins that bear a classical nuclear localization sequence (cNLS). These cargo proteins are linked to the major nuclear protein import factor, importin‐β, by the importin‐α adapter, after which cargo/carrier complexes enter the nucleus through nuclear pores. In the nucleus, cargo is released by the action of RanGTP and the nuclear pore protein Nup2, after which the importins are recycled to the cytoplasm for further transport cycles. The nuclear export of importin‐α is mediated by Cse1/CAS. Here, we exploit structures of functionally important complexes to identify residues that are critical for these interactions and provide insight into how cycles of protein import and recycling of importin‐α occur in vivo using a Saccharomyces cerevisiae model. We examine how these molecular interactions impact protein localization, cargo import, function and complex formation. We show that reversing the charge of key residues in importin‐α (Arg44) or Cse1 (Asp220) results in loss of function of the respective proteins and impairs complex formation both in vitro and in vivo. To extend these results, we show that basic residues in the Nup2 N‐terminus are required for both Nup2 interaction with importin‐α and Nup2 function. These results provide a more comprehensive mechanistic model of how Cse1, RanGTP and Nup2 function in concert to mediate cNLS‐cargo release in the nucleus.     

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。     

Getting on target: the archaeal signal recognition particle

Protein translocation begins with the efficient targeting of secreted and membrane proteins to complexes embedded within the membrane. In Eukarya and Bacteria, this is achieved through the interaction of the signal recognition particle (SRP) with the nascent polypeptide chain. In Archaea, homologs of eukaryal and bacterial SRP-mediated translocation pathway components have been identified. Biochemical analysis has revealed that although the archaeal system incorporates various facets of the eukaryal and bacterial targeting systems, numerous aspects of the archaeal system are unique to this domain of life. Moreover, it is becoming increasingly clear that elucidation of the archaeal SRP pathway will provide answers to basic questions about protein targeting that cannot be obtained from examination of eukaryal or bacterial models. In this review, recent data regarding the molecular composition, functional behavior and evolutionary significance of the archaeal signal recognition particle pathway are discussed.     

Signal sequence- and translation-independent mRNA localization to the endoplasmic reticulum

The process of mRNA localization typically utilizes cis-targeting elements and trans-recognition factors to direct the compartmental organization of translationally suppressed mRNAs. mRNA localization to the endoplasmic reticulum (ER), in contrast, occurs via a co-translational, signal sequence/signal recognition particle (SRP)-dependent mechanism. We have utilized cell fractionation/cDNA microarray analysis, shRNA-mediated suppression of SRP expression, and mRNA reporter construct studies to define the role of the SRP pathway in ER-directed mRNA localization. Cell fractionation studies of mRNA partitioning between the cytosol and ER demonstrated the expected enrichment of cytosolic/nucleoplasmic protein-encoding mRNAs and secretory/integral membrane protein-encoding mRNAs in the cytosol and ER fractions, respectively, and identified a subpopulation of cytosolic/nucleoplasmic protein-encoding mRNAs in the membrane-bound mRNA pool. The latter finding suggests a signal sequence-independent pathway of ER-directed mRNA localization. Extending from these findings, mRNA partitioning was examined in stable SRP54 shRNA knockdown HeLa cell lines. shRNA-directed reductions in SRP did not globally alter mRNA partitioning patterns, although defects in membrane protein processing were observed, further suggesting the existence of multiple pathways for mRNA localization to the ER. ER localization of GRP94-encoding mRNA was observed when translation was disabled by mutation of the start codon/insertion of a 5'UTR stem-loop structure or upon deletion of the encoded signal sequence. Combined, these data indicate that the mRNA localization to the ER can be conferred independent of the signal sequence/SRP pathway and suggest that mRNA localization to the ER may utilize cis-encoded targeting information.     

Expression, purification, and crystallography of the conserved methionine-rich domain of human signal recognition particle 54 kDa protein.

Protein SRP54 is an essential component of eukaryotic signal recognition particle (SRP). The methionine-rich M-domain (SRP54M or 54M) interacts with the SRP RNA and is also involved in the binding to signal peptides of secretory proteins during their targeting to cellular membranes. To gain insight into the molecular details of SRP-mediated protein targeting, we studied the human 54M polypeptide. The recombinant human protein was expressed successfully in Escherichia coli and was purified to homogeneity. Our studies determined the sites that were susceptible to limited proteolysis, with the goal to design smaller functional mutant derivatives that lacked nonessential amino acid residues from both termini. Of the four polypeptides produced by V8 protease or chymotrypsin, 54MM-2 was the shortest (120 residues; Mr = 13,584.8), but still contained the conserved amino acids suggested to associate with the signal peptide or the SRP RNA. 54MM-2 was cloned, expressed, purified to homogeneity, and was shown to bind human SRP RNA in the presence of protein SRP19, indicating that it was functional. Highly reproducible conditions for the crystallization of 54MM-2 were established. Examination of the crystals by X-ray diffraction showed an orthorhombic unit cell of dimensions a = 29.127 A, b = 63.693 A, and c = 129.601 A, in space group P2(1)2(1)2(1), with reflections extending to at least 2.0 A.     

Protein traffic for secretion and related machinery of Bacillus subtilis

Gram-positive sporulating Bacillus subtilis secretes high levels of protein. Its complete genome sequence, published in 1997, encodes 4,106 proteins. Bioinformatic searches have predicted that about half of all B. subtilis proteins are related to the cell membrane through export to the extracellular medium, insertion, and attachment. Key features of the B. subtilis protein secretion machinery are the absence of an Escherichia coli SecB homolog and the presence of an SRP (signal recognition particle) that is structurally rather similar to human SRP. In addition, B. subtilis contains five type I signal peptidases (SipS, T, U, V, and W). Our in vitro assay system indicated that co-operation between the SRP-protein targeting system to the cell membrane and the Sec protein translocation machinery across the cytoplasmic membrane constitutes the major protein secretion pathway in B. subtilis. Furthermore, the function of the SRP-Sec pathway in protein localization to the cell membrane and spore was analyzed.     

Is Ffh required for export of secretory proteins?

In Escherichia coli, protein export from the cytoplasm may occur via the signal recognition particle (SRP)-dependent pathway or the Sec-dependent pathway. Membrane proteins utilize the SRP-dependent route, whereas many secretory proteins use the cytoplasmic Sec machinery. To examine the possibility that signal peptide hydrophobicity governs which targeting route is utilized, we used a series of PhoA signal sequence mutants which vary only by incremental hydrophobicity changes. We show that depletion of SRP, but not trigger factor, affects all the mutants examined. These results suggest secretory proteins with a variety of signal sequences, as well as membrane proteins, require SRP for export.     

SRP-mediated protein targeting: structure and function revisited

The signal recognition particle (SRP) and its membrane-bound receptor (SR) deliver membrane proteins and secretory proteins to the translocation channel in the plasma membrane (or the endoplasmic reticulum). The general outline of the SRP pathway is conserved in all three kingdoms of life. During the past decade, structure determination together with functional studies has brought our understanding of the SRP-mediated protein transport to an almost molecular level. An impressive amount of new information especially on the prokaryotic SRP is integrated into the current picture of the SRP pathway.     

Concerted complex assembly and GTPase activation in the chloroplast signal recognition particle

The universally conserved signal recognition particle (SRP) and SRP receptor (SR) mediate the cotranslational targeting of proteins to cellular membranes. In contrast, a unique chloroplast SRP in green plants is primarily dedicated to the post-translational targeting of light harvesting chlorophyll a/b binding (LHC) proteins. In both pathways, dimerization and activation between the SRP and SR GTPases mediate the delivery of cargo; whether and how the GTPase cycle in each system adapts to its distinct substrate proteins were unclear. Here, we show that interactions at the active site essential for GTPase activation in the chloroplast SRP and SR play key roles in the assembly of the GTPase complex. In contrast to their cytosolic homologues, GTPase activation in the chloroplast SRP-SR complex contributes marginally to the targeting of LHC proteins. These results demonstrate that complex assembly and GTPase activation are highly coupled in the chloroplast SRP and SR and suggest that the chloroplast GTPases may forego the GTPase activation step as a key regulatory point. These features may reflect adaptations of the chloroplast SRP to the delivery of their unique substrate protein.     

Isolation and characterization of an Arabidopsis thaliana gene for the 54 kDa subunit of the signal recognition particle

The first step in the routing of newly synthesized proteins into the secretory pathway is the binding of the nascent signal sequence to the signal recognition particle. The mammalian signal recognition particle is a complex consisting of 6 proteins and a single 7S RNA molecule. Signal recognition particle-like complexes have been described from wheat and maize but none of the protein components have yet been described from any plant species. Here we report the cloning and characterization of an Arabidopsis thaliana gene encoding the 54 kDa protein subunit of the signal recognition particle. This is the first report of a SRP-54 sequence for any plant species and the first genomic sequence for any multicellular organism.Abbreviations ER endoplasmic reticulum - PCR polymerase chain reaction - SRP signal recognition particle