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PubMed1996

The TSR1 gene of Yarrowia lipolytica is involved in the signal recognition particle-dependent translocation pathway of secretory proteins.

We have isolated suppressors (tsr1 to tsr5) of the thermosensitive growth of the scr2.II-13 mutation, which affects the stability of the signal recognition particle. The growth of these mutants is largely affected in the SCR2 context at 34 degrees C. We have studied the synthesis and secretion of an alkaline extracellular protease (AEP) in both wild-type and tsr1-1(SCR2(+)) thermosensitive mutant strains. Pulse-chase labeling and immunoprecipitation of this protein showed that the level of AEP precursors in the tsr1-1(SCR2(+)) strain is 70% less than in the wild-type strain under conditions where the global protein synthesis is practically unaffected. This defect was observed as early as 10 min after the shift to nonpermissive temperature. In neither strain was there any effect on the kinetics of secretion, and no cytoplasmic accumulation was detected. We have cloned the TSR1 gene by complementing the thermosensitive phenotype of a tsr1-1(SCR2(+)) mutant. Analysis of the TSR1 DNA sequence revealed an open reading frame of 1383 base pairs, encoding a serine-rich protein of 461 amino acids with an amino-terminal signal peptide, and a membrane-spanning domain of 20 amino acids that could act as a stop transfer signal to ensure membrane localization of Tsr1p. Two homologues of the TSR1 gene were identified in Saccharomyces cerevisiae (YHC8) and Hansenula polymorpha (YLU2). Disruption of the TSR1 gene revealed that it is an essential single-copy gene. The TSR1 gene encodes a single mRNA of 1.5 kilobase pairs. The study of the synthesis and secretion of AEP in the complemented tsr1-1(SCR2(+),TSR1(+)) strain revealed that the TSR1 gene ensures complete recovery of the synthesis defect and thus could encode an important component of the endoplasmic reticulum membrane involved in the early steps of the signal recognition particle-dependent translocation pathway.

Amino Acid Sequence↗
PubMed1986

Signal recognition particle-dependent membrane insertion of mouse invariant chain: a membrane-spanning protein with a cytoplasmically exposed amino terminus.

Invariant (Ii) chain is a membrane-spanning protein that is found associated intracellularly with class II histocompatibility antigens. In the endoplasmic reticulum Ii chain spans the membrane and exposes the NH2 terminus on the cytoplasmic and the COOH terminus on the lumenal side. This orientation across the membrane is demonstrated directly with the monoclonal antibody In-1, which exclusively recognizes the NH2 terminal cytoplasmically exposed part of Ii chain. Membrane insertion of Ii chain requires signal recognition particle and docking protein. When tested in a wheat germ cell free system, signal recognition particle arrests translation of Ii chain. No signal sequence is cleaved from Ii chain upon membrane insertion.

Amino Acid Sequence↗
PubMed2004

SRP meets the ribosome.

Cotranslational targeting directly couples synthesis of proteins to their translocation across or insertion into membranes. The signal recognition particle (SRP) and its membrane-bound receptor facilitate the targeting of the translation machinery, the ribosome, via recognition of a signal sequence in the nascent peptide chain. By combining structures of free and ribosome-bound SRP we derive a structural model describing the dynamic nature of SRP when it meets the ribosome.

Animals↗
PubMed1997

Yarrowia lipolytica TSR1 gene product. A novel endoplasmic reticulum membrane component involved in the signal recognition particle-dependent translocation pathway.

The tsr1-1 mutation has been initially identified as an extragenic suppressor of the scr2.II-13 mutation that alters the 7SL RNA component of the signal recognition particle (SRP) and results in severe defects in protein translocation and SRP stability. We showed previously that the TSR1 gene was essential and that the tsr1-1 mutation allowed complete recovery of scr2.II-13-associated secretory defects. We show here that the tsr1-1 mutation also restores SRP stability in an scr2.II-13 context. The TSR1 gene product (Tsr1p) is stably associated with rapidly sedimenting material and cofractionates with the lumenal protein Kar2p of the endoplasmic reticulum; it behaves in protease protection assays as a transmembrane component. Coimmunoprecipitation experiments revealed a physical interaction with Kar2p and with ribosomal components associated to the 5.8S rRNA as well as with SRP components like Sec65p and 7SL RNA. We propose that Tsr1p is an important component of the endoplasmic reticulum membrane, interacting both with the SRP-ribosome complex in the cytosol and with Kar2p in the lumen of the endoplasmic reticulum.

Biological Transport↗
PubMed2002

Classification and evolution of P-loop GTPases and related ATPases.

Sequences and available structures were compared for all the widely distributed representatives of the P-loop GTPases and GTPase-related proteins with the aim of constructing an evolutionary classification for this superclass of proteins and reconstructing the principal events in their evolution. The GTPase superclass can be divided into two large classes, each of which has a unique set of sequence and structural signatures (synapomorphies). The first class, designated TRAFAC (after translation factors) includes enzymes involved in translation (initiation, elongation, and release factors), signal transduction (in particular, the extended Ras-like family), cell motility, and intracellular transport. The second class, designated SIMIBI (after signal recognition particle, MinD, and BioD), consists of signal recognition particle (SRP) GTPases, the assemblage of MinD-like ATPases, which are involved in protein localization, chromosome partitioning, and membrane transport, and a group of metabolic enzymes with kinase or related phosphate transferase activity. These two classes together contain over 20 distinct families that are further subdivided into 57 subfamilies (ancient lineages) on the basis of conserved sequence motifs, shared structural features, and domain architectures. Ten subfamilies show a universal phyletic distribution compatible with presence in the last universal common ancestor of the extant life forms (LUCA). These include four translation factors, two OBG-like GTPases, the YawG/YlqF-like GTPases (these two subfamilies also consist of predicted translation factors), the two signal-recognition-associated GTPases, and the MRP subfamily of MinD-like ATPases. The distribution of nucleotide specificity among the proteins of the GTPase superclass indicates that the common ancestor of the entire superclass was a GTPase and that a secondary switch to ATPase activity has occurred on several independent occasions during evolution. The functions of most GTPases that are traceable to LUCA are associated with translation. However, in contrast to other superclasses of P-loop NTPases (RecA-F1/F0, AAA+, helicases, ABC), GTPases do not participate in NTP-dependent nucleic acid unwinding and reorganizing activities. Hence, we hypothesize that the ancestral GTPase was an enzyme with a generic regulatory role in translation, with subsequent diversification resulting in acquisition of diverse functions in transport, protein trafficking, and signaling. In addition to the classification of previously known families of GTPases and related ATPases, we introduce several previously undetected families and describe new functional predictions.

Adenosine Triphosphatases↗
PubMed1998

Signal recognition particle-dependent targeting of ribosomes to the rough endoplasmic reticulum in the absence and presence of the nascent polypeptide-associated complex.

Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC), respectively, act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP-dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the nonselective interaction between the ribosome and the Sec61 complex.

Animals↗
PubMed1989

Nascent secretory chain binding and translocation are distinct processes: differentiation by chemical alkylation.

We have investigated the effects of chemical alkylation of microsomal membranes on nascent chain binding and translocation. Assays were conducted using either full-length or truncated preprolactin transcripts in combination with a reconstituted membrane system consisting of proteolyzed rough microsomes and the cytoplasmic domain of the signal recognition particle receptor. Treatment of rough microsomes with N-ethylmaleimide was observed to inhibit preprolactin processing at a site other than the signal recognition particle or the signal recognition particle receptor. As formation of a translocation competent junction between the ribosome/nascent chain complex and the membrane has recently been demonstrated to require GTP (Connolly, T., and R. Gilmore. J. Cell Biol. 1986. 103:2253-2261), the effects of membrane alkylation on this parameter were assessed. N-ethylmaleimide treatment did not inhibit nascent chain targeting or GTP-dependent signal sequence insertion. Translocation of the targeted and inserted nascent chain was, however, blocked. These data indicate (a) that the process of nascent chain translocation is distinct from targeting and signal sequence insertion, and (b) translocation of the peptide chain across the membrane is mediated by an N-ethylmaleimide-sensitive membrane protein component(s). To further substantiate the observation that nascent chain targeting and signal sequence insertion can be distinguished from translocation, the temperature dependencies of the two phenomena were compared. Signal sequence insertion occurred at low temperatures (4 degrees C) and was maximal between 10 and 15 degrees C. Translocation was only observed at higher temperatures and was maximal between 25 and 30 degrees C.

Alkylation↗
PubMed2001

Multifaceted physiological response allows yeast to adapt to the loss of the signal recognition particle-dependent protein-targeting pathway.

Translational control has recently been recognized as an important facet of adaptive responses to various stress conditions. We describe the adaptation response of the yeast Saccharomyces cerevisiae to the loss of one of two mechanisms to target proteins to the secretory pathway. Using inducible mutants that block the signal recognition particle (SRP) pathway, we find that cells demonstrate a physiological response to the loss of the SRP pathway that includes specific changes in global gene expression. Upon inducing the loss of the SRP pathway, SRP-dependent protein translocation is initially blocked, and cell growth is considerably slowed. Concomitantly, gene expression changes include the induction of heat shock genes and the repression of protein synthesis genes. Remarkably, within hours, the efficiency of protein sorting improves while cell growth remains slow in agreement with the persistent repression of protein synthesis genes. Our results suggest that heat shock gene induction serves to protect cells from mislocalized precursor proteins in the cytosol, whereas reduced protein synthesis helps to regain efficiency in protein sorting by reducing the load on the protein translocation apparatus. Thus, we suggest that cells trade speed in cell growth for fidelity in protein sorting to adjust to life without SRP.

Adaptation, Physiological↗
PubMed2006

Signal recognition particle-dependent inner membrane targeting of the PulG Pseudopilin component of a type II secretion system.

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The beta-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.

Amino Acid Sequence↗
PubMed1999

The signal recognition particle-targeting pathway does not necessarily deliver proteins to the sec-translocase in Escherichia coli.

ProW is an Escherichia coli inner membrane protein that consists of a 100-residue-long periplasmic N-terminal tail (N-tail) followed by seven closely spaced transmembrane segments. N-tail translocation presumably proceeds in a C-to-N-terminal direction and represents a poorly understood aspect of membrane protein biogenesis. Here, using an in vivo depletion approach, we show that N-tail translocation in a ProW derivative comprising the N-tail and the first transmembrane segment fused to the globular P2 domain of leader peptidase depends both on the bacterial signal recognition particle (SRP) and the Sec-translocase. Surprisingly, however, a deletion construct with only one transmembrane segment downstream of the N-tail can assemble properly even under severe depletion of SecE, a central component of the Sec-translocase, but not under SRP-depletion conditions. To our knowledge, this is the first demonstration that the SRP-targeting pathway does not necessarily deliver SRP-dependent inner membrane proteins to the Sec-translocase. The data further suggest that N-tail translocation can proceed in the absence of a functional Sec-translocase.

ATP-Binding Cassette Transporters↗
PubMed2006

Export of the pseudopilin XcpT of the Pseudomonas aeruginosa type II secretion system via the signal recognition particle-Sec pathway.

Type IV pilins and pseudopilins are found in various prokaryotic envelope protein complexes, including type IV pili and type II secretion machineries of gram-negative bacteria, competence systems of gram-positive bacteria, and flagella and sugar-binding structures in members of the archaeal kingdom. The precursors of these proteins have highly conserved N termini, consisting of a short, positively charged leader peptide, which is cleaved off by a dedicated peptidase during maturation, and a hydrophobic stretch of approximately 20 amino acid residues. Which pathway is involved in the inner membrane translocation of these proteins is unknown. We used XcpT, the major pseudopilin from the type II secretion machinery of Pseudomonas aeruginosa, as a model to study this process. Transport of an XcpT-PhoA hybrid was shown to occur in the absence of other Xcp components in P. aeruginosa and in Escherichia coli. Experiments with conditional sec mutants and reporter-protein fusions showed that this transport process involves the cotranslational signal recognition particle targeting route and is dependent on a functional Sec translocon.

Adenosine Triphosphatases↗
PubMed1989

Parallel effects of signal peptide hydrophobic core modifications on co-translational translocation and post-translational cleavage by purified signal peptidase.

The length of the hydrophobic core of the bovine parathyroid hormone signal peptide was modified by in vitro mutagenesis. Extension of the hydrophobic core by three amino acids at the NH2-terminal end had little effect on the proteolytic processing of the signal peptide by microsomal membranes. Deletion of 6 of the 12 amino acids in the core eliminated translocation and processing of the modified protein. Deletion of pairs of amino acids across the core resulted in position-dependent inhibition of signal activity unrelated to hydrophobicity but inversely related to the hydrophobic moments of the modified cores. Deletions in the NH2-terminal region of the core were strongly inhibitory for proteolytic processing whereas deletions in the COOH-terminal region had no effect or increased processing when assessed either co-translationally with microsomal membranes or post-translationally with purified hen oviduct signal peptidase. Deletion of cysteine 18 and alanine 19 increased processing, but deletion of cysteine alone or substitution of leucine for cysteine did not increase processing more than deletion of both residues at 18 and 19. Translations of the translocation-defective mutants with pairs of amino acids deleted in a wheat germ system were inhibited by addition of exogenous signal recognition particle suggesting that interactions of the modified signal peptides with signal recognition particle were normal. The position-dependent effects of the hydrophobic core modifications indicate that structural properties of the core in addition to hydrophobicity are important for signal activity. The parallel effects of the modifications on co-translational translocation and post-translational processing by purified signal peptidase suggest that proteins in the signal peptidase complex might be part of, or intimately associated with, membrane proteins involved in the translocation. A model is proposed in which the NH2-terminal region of the hydrophobic core binds to one subunit of the signal peptidase while the other subunit catalyzes the cleavage.

Amino Acid Sequence↗
PubMed2006

Trigger factor binding to ribosomes with nascent peptide chains of varying lengths and sequences.

Trigger factor (TF) is the first protein-folding chaperone to interact with a nascent peptide chain as it emerges from the ribosome. Here, we have used a spin down assay to estimate the affinities for the binding of TF to ribosome nascent chain complexes (RNCs) with peptides of varying lengths and sequences. An in vitro system for protein synthesis assembled from purified Escherichia coli components was used to produce RNCs stalled on truncated mRNAs. The affinity of TF to RNCs exposing RNA polymerase sequences increased with the length of the nascent peptides. TF bound to RNA polymerase RNCs with significantly higher affinity than to inner membrane protein leader peptidase and bacterioopsin RNCs. The latter two RNCs are substrates for signal recognition particle, suggesting complementary affinities of TF and signal recognition particle to nascent peptides targeted for cytoplasm and membrane.

Amino Acid Sequence↗
PubMed2007

X-ray structure of the T. aquaticus FtsY:GDP complex suggests functional roles for the C-terminal helix of the SRP GTPases.

FtsY and Ffh are structurally similar prokaryotic Signal Recognition Particle GTPases that play an essential role in the Signal Recognition Particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The two GTPases assemble in a GTP-dependent manner to form a heterodimeric SRP targeting complex. We report here the 2.1 A X-ray structure of FtsY from T. aquaticus bound to GDP. The structure of the monomeric protein reveals, unexpectedly, canonical binding interactions for GDP. A comparison of the structures of the monomeric and complexed FtsY NG GTPase domain suggests that it undergoes a conformational change similar to that of Ffh NG during the assembly of the symmetric heterodimeric complex. However, in contrast to Ffh, in which the C-terminal helix shifts independently of the other subdomains, the C-terminal helix and N domain of T. aquaticus FtsY together behave as a rigid body during assembly, suggesting distinct mechanisms by which the interactions of the NG domain "module" are regulated in the context of the two SRP GTPases.

Bacterial Proteins↗
PubMed2004

Membrane binding of SRP pathway components in the halophilic archaea Haloferax volcanii.

Across evolution, the signal recognition particle pathway targets extra-cytoplasmic proteins to membranous translocation sites. Whereas the pathway has been extensively studied in Eukarya and Bacteria, little is known of this system in Archaea. In the following, membrane association of FtsY, the prokaryal signal recognition particle receptor, and SRP54, a central component of the signal recognition particle, was addressed in the halophilic archaea Haloferax volcanii. Purified H. volcanii FtsY, the FtsY C-terminal GTP-binding domain (NG domain) or SRP54, were combined separately or in different combinations with H. volcanii inverted membrane vesicles and examined by gradient floatation to differentiate between soluble and membrane-bound protein. Such studies revealed that both FtsY and the FtsY NG domain bound to H. volcanii vesicles in a manner unaffected by proteolytic pretreatment of the membranes, implying that in Archaea, FtsY association is mediated through the membrane lipids. Indeed, membrane association of FtsY was also detected in intact H. volcanii cells. The contribution of the NG domain to FtsY binding in halophilic archaea may be considerable, given the low number of basic charges found at the start of the N-terminal acidic domain of haloarchaeal FtsY proteins (the region of the protein thought to mediate FtsY-membrane association in Bacteria). Moreover, FtsY, but not the NG domain, was shown to mediate membrane association of H. volcanii SRP54, a protein that did not otherwise interact with the membrane.

Amino Acid Sequence↗
PubMed2005

Double-spanning plant viral movement protein integration into the endoplasmic reticulum membrane is signal recognition particle-dependent, translocon-mediated, and concerted.

The current model for cell-to-cell movement of plant viruses holds that transport requires virus-encoded movement proteins that intimately associate with endoplasmic reticulum membranes. We have examined the early stages of the integration into endoplasmic reticulum membranes of a double-spanning viral movement protein using photocross-linking. We have discovered that this process is cotranslational and proceeds in a signal recognition particle-dependent manner. In addition, nascent chain photocross-linking to Sec61alpha and translocating chain-associated membrane protein reveal that viral membrane protein insertion takes place via the translocon, as with most eukaryotic membrane proteins, but that the two transmembrane segments of the viral protein leave the translocon and enter the lipid bilayer together.

Calcium-Binding Proteins↗
PubMed1998

A mutation in the Escherichia coli secY gene that produces distinct effects on inner membrane protein insertion and protein export.

E. coli strains that contain the secY40 mutation are cold-sensitive, but protein export defects have not been observed even at the nonpermissive temperature. Here we describe experiments designed to explain the conditional phenotype associated with this allele. We found that combining the secY40 mutation with defects in the signal recognition particle targeting pathway led to synthetic lethality. Since the signal recognition particle is required for the insertion of inner membrane proteins (IMPs) into the cytoplasmic membrane but not for protein export, this observation prompted us to examine the effect of the secY40 mutation on IMP biogenesis. The membrane insertion of all IMPs that we tested was impaired at both permissive and nonpermissive temperatures in secY40 cells grown in either rich or minimal medium. The magnitude of the insertion defects was greatest in cells grown at low temperature in rich medium, conditions in which the growth defect was most pronounced. Consistent with previous reports, we could not detect protein export defects in secY40 cells grown in minimal medium. Upon growth in rich medium, only slight protein export defects were observed. Taken together, these results suggest that the impairment of IMP insertion causes the cold sensitivity of secY40 strains. Furthermore, these results provide the first evidence that the protein export and membrane protein insertion functions of the translocon are genetically separable.

Bacterial Proteins↗