Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains

Most proteins exported from mammalian cells contain a signal sequence which mediates targeting to and insertion into the membrane of the endoplasmic reticulum (ER). Involved in this process are the signal-recognition particle (SRP) and docking protein (DP), the receptor for SRP in the ER membrane. SRP interacts with the signal sequence on nascent polypeptide chains and retards their further elongation, which resumes only after interaction of the arrested ribosomal complex with the docking protein. SRP is a ribonucleoprotein particle comprising a 7S RNA and six polypeptides with relative molecular masses (Mr) of 9,000 (9K) 14K, 19K, 54K, 68K and 72K (ref. 1). The 9K and 14K proteins are essential for elongation arrest and the 68K-72K heterodimer is required for docking to the ER membrane. The 54K protein binds to the signal sequence when it emerges from the ribosome. Docking protein consists of two polypeptides, a 72K alpha-subunit (DP alpha) and a 30K beta-subunit (DP beta). No components structurally homologous to SRP and docking protein have yet been found in yeast or Escherichia coli. To understand the molecular nature of the interaction between the signal sequence and its receptor(s) we have characterized a complementary DNA coding for the 54K protein of SRP. Significant sequence homology was found to part of DP alpha and two E. coli proteins of unknown function. The homologous region includes a putative GTP-binding domain.     

A signal sequence receptor in the endoplasmic reticulum membrane

Protein translocation across the endoplasmic reticulum (ER) membrane is triggered at several stages by information contained in the signal sequence. Initially, the signal sequence of a nascent secretory protein upon emergence from the ribosome is recognized by a polypeptide of relative molecular mass 54,000 (Mr54K) which is part of the signal recognition particle (SRP). Binding of SRP may induce a site-specific elongation arrest of translation in vitro. Attachment of the arrested translation complex to the ER membrane is mediated by the SRP-receptor (docking protein) and is accompanied by displacement of the SRP from both the ribosome and the signal sequence. We have investigated the fate of the signal sequence following the disengagement of SRP and its receptor by a crosslinking approach. We report here that the signal sequence of nascent preprolactin, after its release from the SRP, interacts with a newly discovered component, a signal sequence receptor (SSR), which is an integral, glycosylated protein of the rough ER membrane (Mr approximately 35K).     

Structure of the E. coli signal recognition particle bound to a translating ribosome

The prokaryotic signal recognition particle (SRP) targets membrane proteins into the inner membrane. It binds translating ribosomes and screens the emerging nascent chain for a hydrophobic signal sequence, such as the transmembrane helix of inner membrane proteins. If such a sequence emerges, the SRP binds tightly, allowing the SRP receptor to lock on. This assembly delivers the ribosome-nascent chain complex to the protein translocation machinery in the membrane. Using cryo-electron microscopy and single-particle reconstruction, we obtained a 16 A structure of the Escherichia coli SRP in complex with a translating E. coli ribosome containing a nascent chain with a transmembrane helix anchor. We also obtained structural information on the SRP bound to an empty E. coli ribosome. The latter might share characteristics with a scanning SRP complex, whereas the former represents the next step: the targeting complex ready for receptor binding. High-resolution structures of the bacterial ribosome and of the bacterial SRP components are available, and their fitting explains our electron microscopic density. The structures reveal the regions that are involved in complex formation, provide insight into the conformation of the SRP on the ribosome and indicate the conformational changes that accompany high-affinity SRP binding to ribosome nascent chain complexes upon recognition of the signal sequence.     

Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle

Protein targeting to the endoplasmic reticulum in mammalian cells is catalysed by signal recognition particle (SRP). Cross-linking experiments have shown that the subunit of relative molecular mass 54,000 (Mr 54K; SRP54) interacts directly with signal sequences as they emerge from the ribosome. Here we present the sequence of a complementary DNA clone of SRP54 which predicts a protein that contains a putative GTP-binding domain and an unusually methionine-rich domain. The properties of this latter domain suggest that it contains the signal sequence binding site. A previously uncharacterized Escherichia coli protein has strong homology to both domains. Closely homologous GTP-binding domains are also found in the alpha-subunit of the SRP receptor (SR alpha, docking protein) in the endoplasmic reticulum membrane and in a second E. coli protein, ftsY, which resembles SR alpha. Recent work has shown that SR alpha is a GTP-binding protein and that GTP is required for the release of SRP from the signal sequence and the ribosome on targeting to the endoplasmic reticulum membrane. We propose that SRP54 and SR alpha use GTP in sequential steps of the targeting reaction and that essential features of such a pathway are conserved from bacteria to mammals.     

YidC mediates membrane protein insertion in bacteria

The basic machinery for the translocation of proteins into or across membranes is remarkably conserved from Escherichia coli to humans. In eukaryotes, proteins are inserted into the endoplasmic reticulum using the signal recognition particle (SRP) and the SRP receptor, as well as the integral membrane Sec61 trimeric complex (composed of alpha, beta and gamma subunits). In bacteria, most proteins are inserted by a related pathway that includes the SRP homologue Ffh, the SRP receptor FtsY, and the SecYEG trimeric complex, where Y and E are related to the Sec61 alpha and gamma subunits, respectively. Proteins in bacteria that exhibit no dependence on the Sec translocase were previously thought to insert into the membrane directly without the aid of a protein machinery. Here we show that membrane insertion of two Sec-independent proteins requires YidC. YidC is essential for E. coli viability and homologues are present in mitochondria and chloroplasts. Depletion of YidC also interferes with insertion of Sec-dependent membrane proteins, but it has only a minor effect on the export of secretory proteins. These results provide evidence for an additional component of the translocation machinery that is specialized for the integration of membrane proteins.     

Structure of the SRP19 RNA complex and implications for signal recognition particle assembly

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein. It associates with ribosomes to mediate co-translational targeting of membrane and secretory proteins to biological membranes. In mammalian cells, the SRP consists of a 7S RNA and six protein components. The S domain of SRP comprises the 7S.S part of RNA bound to SRP19, SRP54 and the SRP68/72 heterodimer; SRP54 has the main role in recognizing signal sequences of nascent polypeptide chains and docking SRP to its receptor. During assembly of the SRP, binding of SRP19 precedes and promotes the association of SRP54 (refs 4, 5). Here we report the crystal structure at 2.3 A resolution of the complex formed between 7S.S RNA and SRP19 in the archaeon Methanococcus jannaschii. SRP19 bridges the tips of helices 6 and 8 of 7S.S RNA by forming an extensive network of direct protein RNA interactions. Helices 6 and 8 pack side by side; tertiary RNA interactions, which also involve the strictly conserved tetraloop bases, stabilize helix 8 in a conformation competent for SRP54 binding. The structure explains the role of SRP19 and provides a molecular framework for SRP54 binding and SRP assembly in Eukarya and Archaea.     

The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle

Hydrophobic signal sequences direct the translocation of nascent secretory proteins and many membrane proteins across the membrane of the endoplasmic reticulum. Initiation of this process involves the signal recognition particle (SRP), which consists of six polypeptide chains and a 7S RNA and interacts with ribosomes carrying nascent secretory polypeptide chains. In the case of aminoterminal, cleavable signal sequences, in the absence of microsomal membranes it exerts a site-specific translational arrest in vitro. The size of the arrested fragment (60-70 amino-acid residues) suggests that elongation stops when the signal sequence has emerged fully from the ribosome. However, a direct interaction between the signal sequence and SRP has not previously been demonstrated and has even been questioned recently. We now show for the first time a direct interaction between the signal sequence of a secretory protein and a component of SRP, the 45K polypeptide (relative molecular mass (Mr) 54,000). This was achieved by means of a new method of affinity labelling which involves the translational incorporation of an amino acid, carrying a photoreactive group, into nascent polypeptides.     

Structure of the signal recognition particle interacting with the elongation-arrested ribosome

Cotranslational translocation of proteins across or into membranes is a vital process in all kingdoms of life. It requires that the translating ribosome be targeted to the membrane by the signal recognition particle (SRP), an evolutionarily conserved ribonucleoprotein particle. SRP recognizes signal sequences of nascent protein chains emerging from the ribosome. Subsequent binding of SRP leads to a pause in peptide elongation and to the ribosome docking to the membrane-bound SRP receptor. Here we present the structure of a targeting complex consisting of mammalian SRP bound to an active 80S ribosome carrying a signal sequence. This structure, solved to 12 A by cryo-electron microscopy, enables us to generate a molecular model of SRP in its functional conformation. The model shows how the S domain of SRP contacts the large ribosomal subunit at the nascent chain exit site to bind the signal sequence, and that the Alu domain reaches into the elongation-factor-binding site of the ribosome, explaining its elongation arrest activity.     

Chicken ovalbumin contains an internal signal sequence.

Ovalbumin is shown to contain an internal rather than an amino-terminal signal sequence. This internal signal can be recovered in a tryptic fragment which comprises residues 229--276 of mature ovalbumin and contains a region of striking sequence homology to amino-terminal cleaved signals of two other oviduct secretory proteins. The isolated tryptic fragment is used as a probe to demonstrate that a signal receptor is present in membrane vesicles derived from the rough but not in those derived from the smooth endoplasmic reticulum.     

The S. cerevisiae SEC65 gene encodes a component of yeast signal recognition particle with homology to human SRP19.

Translocation of proteins across the endoplasmic reticulum (ER) membrane represents the first step in the eukaryotic secretory pathway. In mammalian cells, the targeting of secretory and membrane protein precursors to the ER is mediated by signal recognition particle (SRP), a cytosolic ribonucleoprotein complex comprising a molecule of 7SL RNA and six polypeptide subunits (relative molecular masses 9, 14, 19, 54, 68 and 72K). In Saccharomyces cerevisiae, a homologue of the 54K subunit (SRP54) co-purifies with a small cytoplasmic RNA, scR1 (refs 4, 5). Genetic data indicate that SRP54 and scR1 are involved in translocation in vivo, suggesting the existence of an SRP-like activity in yeast. Whether this activity requires additional components similar to those found in mammalian SRP is not known. We have recently reported a genetic selection that led to the isolation of a yeast mutant, sec65-1, which is conditionally defective in the insertion of integral membrane proteins into the ER. Here we report the cloning and sequencing of the SEC65 gene, which encodes a 31.2K protein with significant sequence similarity to the 19K subunit of human SRP (SRP19). We also report the cloning of a multicopy suppressor of sec65-1, and its identification as the previously defined SRP54 gene, providing genetic evidence for an interaction between these gene products in vivo.     

Following the signal sequence from ribosomal tunnel exit to signal recognition particle

Membrane and secretory proteins can be co-translationally inserted into or translocated across the membrane. This process is dependent on signal sequence recognition on the ribosome by the signal recognition particle (SRP), which results in targeting of the ribosome-nascent-chain complex to the protein-conducting channel at the membrane. Here we present an ensemble of structures at subnanometre resolution, revealing the signal sequence both at the ribosomal tunnel exit and in the bacterial and eukaryotic ribosome-SRP complexes. Molecular details of signal sequence interaction in both prokaryotic and eukaryotic complexes were obtained by fitting high-resolution molecular models. The signal sequence is presented at the ribosomal tunnel exit in an exposed position ready for accommodation in the hydrophobic groove of the rearranged SRP54 M domain. Upon ribosome binding, the SRP54 NG domain also undergoes a conformational rearrangement, priming it for the subsequent docking reaction with the NG domain of the SRP receptor. These findings provide the structural basis for improving our understanding of the early steps of co-translational protein sorting.     

Signal-sequence recognition by an Escherichia coli ribonucleoprotein complex.

Hydrophobic signal-sequences direct the transfer of secretory proteins across the inner membrane of prokaryotes and the endoplasmic reticulum membranes of eukaryotes. In mammalian cells, signal-sequences are recognized by the 54K protein (M(r) 54,000) of the signal recognition particle (SRP) which is believed to hold the nascent chain in a translocation-competent conformation until it contacts the endoplasmic reticulum membrane. The SRP consists of a 7S RNA and six different polypeptides. The 7S RNA and the 54K signal-sequence-binding protein (SRP54) of mammalian SRP exhibit strong sequence similarity to the 4.5S RNA and P48 protein (Ffh) of Escherichia coli which form a ribonucleoprotein particle. Depletion of 4.5S RNA or overproduction of P48 causes the accumulation of the beta-lactamase precursor, although not of other secretory proteins. Whether 4.5S RNA and P48 are part of an SRP-like complex with a role in protein export is controversial. Here we show that the P48/4.5S RNA ribonucleoprotein complex interacts specifically with the signal sequence of a nascent secretory protein and therefore is a signal recognition particle.     

Substrate twinning activates the signal recognition particle and its receptor

Signal sequences target proteins for secretion from cells or for integration into cell membranes. As nascent proteins emerge from the ribosome, signal sequences are recognized by the signal recognition particle (SRP), which subsequently associates with its receptor (SR). In this complex, the SRP and SR stimulate each other's GTPase activity, and GTP hydrolysis ensures unidirectional targeting of cargo through a translocation pore in the membrane. To define the mechanism of reciprocal activation, we determined the 1.9 A structure of the complex formed between these two GTPases. The two partners form a quasi-two-fold symmetrical heterodimer. Biochemical analysis supports the importance of the extensive interaction surface. Complex formation aligns the two GTP molecules in a symmetrical, composite active site, and the 3'OH groups are essential for association, reciprocal activation and catalysis. This unique circle of twinned interactions is severed twice on hydrolysis, leading to complex dissociation after cargo delivery.     

Structural basis for semaphorin signalling through the plexin receptor

Semaphorins and their receptor plexins constitute a pleiotropic cell-signalling system that is used in a wide variety of biological processes, and both protein families have been implicated in numerous human diseases. The binding of soluble or membrane-anchored semaphorins to the membrane-distal region of the plexin ectodomain activates plexin's intrinsic GTPase-activating protein (GAP) at the cytoplasmic region, ultimately modulating cellular adhesion behaviour. However, the structural mechanism underlying the receptor activation remains largely unknown. Here we report the crystal structures of the semaphorin 6A (Sema6A) receptor-binding fragment and the plexin A2 (PlxnA2) ligand-binding fragment in both their pre-signalling (that is, before binding) and signalling (after complex formation) states. Before binding, the Sema6A ectodomain was in the expected 'face-to-face' homodimer arrangement, similar to that adopted by Sema3A and Sema4D, whereas PlxnA2 was in an unexpected 'head-on' homodimer arrangement. In contrast, the structure of the Sema6A-PlxnA2 signalling complex revealed a 2:2 heterotetramer in which the two PlxnA2 monomers dissociated from one another and docked onto the top face of the Sema6A homodimer using the same interface as the head-on homodimer, indicating that plexins undergo 'partner exchange'. Cell-based activity measurements using mutant ligands/receptors confirmed that the Sema6A face-to-face dimer arrangement is physiologically relevant and is maintained throughout signalling events. Thus, homodimer-to-heterodimer transitions of cell-surface plexin that result in a specific orientation of its molecular axis relative to the membrane may constitute the structural mechanism by which the ligand-binding 'signal' is transmitted to the cytoplasmic region, inducing GAP domain rearrangements and activation.     

SEC65 gene product is a subunit of the yeast signal recognition particle required for its integrity.

Protein targeting to the endoplasmic reticulum (ER) in mammalian cells is catalysed by the signal recognition particle (SRP), which consists of six protein subunits and an RNA subunit. Saccharomyces cerevisiae SRP is a 16S particle, of which only two subunits have been identified: a protein subunit, SRP54p, which is homologous to the mammalian SRP54 subunit, and an RNA subunit, scR1 (ref. 3). The sec65-1 mutant yeast cells are temperature-sensitive for growth and defective in the translocation of several secreted and membrane-bound proteins. The DNA sequence of the SEC65 gene suggests that its product is related to mammalian SRP19 subunit and may have a similar function. Here we show that SEC65p is a subunit of the S. cerevisiae SRP and that it is required for the stable association of another subunit, SRP54p, with SRP. Overexpression of SRP54p suppresses both growth and protein translocation defects in sec65-1 mutant cells.     

Structure and assembly of the Alu domain of the mammalian signal recognition particle

The Alu domain of the mammalian signal recognition particle (SRP) comprises the heterodimer of proteins SRP9 and SRP14 bound to the 5' and 3' terminal sequences of SRP RNA. It retards the ribosomal elongation of signal-peptide-containing proteins before their engagement with the translocation machinery in the endoplasmic reticulum. Here we report two crystal structures of the heterodimer SRP9/14 bound either to the 5' domain or to a construct containing both 5' and 3' domains. We present a model of the complete Alu domain that is consistent with extensive biochemical data. SRP9/14 binds strongly to the conserved core of the 5' domain, which forms a U-turn connecting two helical stacks. Reversible docking of the more weakly bound 3' domain might be functionally important in the mechanism of translational regulation. The Alu domain structure is probably conserved in other cytoplasmic ribonucleoprotein particles and retroposition intermediates containing SRP9/14-bound RNAs transcribed from Alu repeats or related elements in genomic DNA.     

An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins

Plasma membrane receptors for hormones, drugs, neurotransmitters and sensory stimuli are coupled to guanine nucleotide regulatory proteins. Recent cloning of the genes and/or cDNAs for several of these receptors including the visual pigment rhodopsin, the adenylate-cyclase stimulatory beta-adrenergic receptor and two subtypes of muscarinic cholinergic receptors has suggested that these are homologous proteins with several conserved structural and functional features. Whereas the rhodopsin gene consists of five exons interrupted by four introns, surprisingly the human and hamster beta-adrenergic receptor genes contain no introns in either their coding or untranslated sequences. We have cloned and sequenced a DNA fragment in the human genome which cross-hybridizes with a full-length beta 2-adrenergic receptor probe at reduced stringency. Like the beta 2-adrenergic receptor this gene appears to be intronless, containing an uninterrupted long open reading frame which encodes a putative protein with all the expected structural features of a G-protein-coupled receptor.     

G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A(2A) adenosine receptor (A(2A)AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A(2A)AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A(2A)AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A(2A)AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active β(2)-adrenergic receptor structure, but locks A(2A)AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.     

Topographical rearrangement of acetylcholine receptors alters channel kinetics

Plasma membranes are dynamic structures of proteins and lipids. Protein-protein or protein-lipid interactions within the membrane are believed to have important roles in many membrane functions, including ion transport, enzyme activity and signal reception. The acetylcholine (ACh) receptor-channel complex in skeletal muscle membrane is one of the best known integral membrane proteins. Its ion transport function is accessible to direct measurement at the single-channel level by the use of the 'giga-seal' patch recording technique. Here we used an in situ electrophoresis technique to rearrange the topography of pre-existing ACh receptor-channels in the muscle membrane, and measured the single-channel kinetics of ACh-activated channels in two different molecular environments within the membrane: those in the diffusely distributed region and those in the ACh receptor clusters induced by the applied field. We found that the channel kinetics are significantly prolonged in the ACh receptor cluster compared with the non-clustered region of the same cell. This result strongly supports the notion that the function of a membrane ionic channel depends on the local molecular environment.