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Results for “Signal Recognition Particle”

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PubMed1992

Random mutagenesis of Schizosaccharomyces pombe SRP RNA: lethal and conditional lesions cluster in presumptive protein binding sites.

Signal recognition particle (SRP), a ribonucleoprotein composed of six polypeptides and one RNA subunit, serves as an adaptor between the cytoplasmic protein synthetic machinery and the translocation apparatus of the endoplasmic reticulum. To begin constructing a functional map of the 7SL RNA component of SRP, we extensively mutagenized the Schizosaccharomyces pombe SRP7 gene. Phenotypes are reported for fifty-two mutant alleles derived from random point mutagenesis, seven alleles created by site-directed mutagenesis to introduce restriction sites into the SRP7 gene, nine alleles designed to pinpoint conditional lesions, and three alleles with extra nucleotides inserted at position 84. Our data indicate that virtually all single nucleotide changes as well as many multiple substitutions in this highly structured RNA are phenotypically silent. Six lethal alleles and eleven which result in sensitivity to the combination of high temperature and elevated osmotic strength were identified. These mutations cluster in conserved regions which, in the mammalian RNA, are protected from nucleolytic agents by SRP proteins. The effects of mutations in the presumptive binding site for a fission yeast SRP 9/14 homolog indicate that both the identity of a conserved residue and the secondary structure within which it is embedded are functionally important. The phenotypes of mutations in Domain IV suggest particular residues as base-specific contacts for the fission yeast SRP54 protein. A single allele which confers temperature-sensitivity in the absence of osmotic perturbants was identified in this study; the growth properties of the mutant strain suggest that the encoded RNA is somewhat defective even at the permissive temperature, and is most likely unable to correctly assemble with SRP proteins at the nonpermissive temperature.

Alleles↗

PubMed2005

Novel autoantibodies against 7SL RNA in patients with polymyositis/dermatomyositis.

OBJECTIVE: Autoantibodies against signal recognition particle (SRP) are detected in patients with polymyositis/dermatomyositis (PM/DM). The SRP consists of 7SL RNA and 6 protein components. We examined autoantibodies against deproteinized 7SL RNA in PM/DM patients with anti-SRP antibodies and evaluated the association of anti-7SL RNA antibodies with PM/DM clinically and serologically. METHODS: Sera from 10 Japanese and 22 North American PM/DM patients with anti-SRP antibodies were tested for the presence of anti-7SL RNA antibodies, using the sera to immunoprecipitate deproteinized RNA extracts derived from HeLa cells. RESULTS: The immunoprecipitation analysis indicated that 5 Japanese (50%) and one North American (5%) patient with anti-SRP antibodies had novel autoantibodies against deproteinized 7SL RNA. The frequency of anti-7SL RNA antibodies was significantly higher in Japanese than North American patients (p = 0.006). The presence of anti-7SL RNA antibodies appeared to be associated with DM (2 patients) and finger swelling (2 PM patients). The seasonal onset of the disease was different (p = 0.008) for Japanese PM/DM patients with anti-7SL RNA antibodies, who developed the disease between October and January (mean month November; p = 0.01) from that of patients without these antibodies, who developed it between June and August (mean month July; p = 0.01). CONCLUSION: Novel autoantibodies against 7SL RNA were identified in patients with PM/DM, and the presence of these antibodies was correlated to ethnic background, clinical features, and season of disease onset. These findings indicated that autoantibodies against 7SL RNA are a novel serological marker for a subset of PM/DM cases.

Adult↗

PubMed2001

Characterization of the sat operon in Streptococcus mutans: evidence for a role of Ffh in acid tolerance.

An essential protein translocation pathway in Escherichia coli and Bacillus subtilis involves the signal recognition particle (SRP), of which the 54-kDa homolog (Ffh) is an essential component. In a previous study, we found that a transposon insertion in the ylxM-ffh intergenic region of the designated secretion and acid tolerance (sat) operon of Streptococcus mutans resulted in an acid-sensitive phenotype. In the present study, we further characterized this genomic region in S. mutans after construction of bona fide sat operon mutants and confirmed the role of the SRP pathway in acid resistance. Northern blot and primer extension analyses identified an acid-inducible promoter upstream of ylxM that was responsible for upregulating the coordinate expression of all five genes of the sat operon when cells were grown at acid pH. Two constitutive promoters, one immediately upstream of satD and one just 3' to the acid-inducible promoter, were also identified. Except for Ffh, the functions of the sat operon gene products are unknown. SatC, SatD, and SatE have no homology to proteins with known functions, although YlxM may function as a transcriptional regulator linked to genes encoding SRP pathway proteins. Nonpolar mutations created in each of the five genes of the sat locus resulted in viable mutants. Most striking, however, was the finding that a mutation in ffh did not result in loss of cell viability, as is the case in all other microbial species in which this pathway has been described. This mutant also lacked immunologically detectable Ffh and was severely affected in resistance to acid. Complementation of the mutation resulted in restoration of acid tolerance and reappearance of cytoplasmic Ffh. These data provide evidence that the SRP pathway plays an important role in acid tolerance in S. mutans.

Bacterial Proteins↗

PubMed2001

Intragenic promoter adaptation and facilitated RNA polymerase III recycling in the transcription of SCR1, the 7SL RNA gene of Saccharomyces cerevisiae.

The SCR1 gene, coding for the 7SL RNA of the signal recognition particle, is the last known class III gene of Saccharomyces cerevisiae that remains to be characterized with respect to its mode of transcription and promoter organization. We show here that SCR1 represents a unique case of a non-tRNA class III gene in which intragenic promoter elements (the TFIIIC-binding A- and B-blocks), corresponding to the D and TpsiC arms of mature tRNAs, have been adapted to a structurally different small RNA without losing their transcriptional function. In fact, despite the presence of an upstream canonical TATA box, SCR1 transcription strictly depends on the presence of functional, albeit quite unusual, A- and B-blocks and requires all the basal components of the RNA polymerase III transcription apparatus, including TFIIIC. Accordingly, TFIIIC was found to protect from DNase I digestion an 80-bp region comprising the A- and B-blocks. B-block inactivation completely compromised TFIIIC binding and transcription capacity in vitro and in vivo. An inactivating mutation in the A-block selectively affected TFIIIC binding to this promoter element but resulted in much more dramatic impairment of in vivo than in vitro transcription. Transcriptional competition and nucleosome disruption experiments showed that this stronger in vivo defect is due to a reduced ability of A-block-mutated SCR1 to compete with other genes for TFIIIC binding and to counteract the assembly of repressive chromatin structures through TFIIIC recruitment. A kinetic analysis further revealed that facilitated RNA polymerase III recycling, far from being restricted to typical small sized class III templates, also takes place on the 522-bp-long SCR1 gene, the longest known class III transcriptional unit.

Amino Acid Sequence↗

PubMed1994

The Srp54 GTPase is essential for protein export in the fission yeast Schizosaccharomyces pombe.

Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein required for targeting a subset of presecretory proteins to the endoplasmic reticulum (ER) membrane. Here we report the results of a series of experiments to define the function of the Schizosaccharomyces pombe homolog of the 54-kDa subunit of mammalian SRP. One-step gene disruption reveals that the Srp54 protein, like SRP RNA, is essential for viability in S. pombe. Precursor to the secretory protein acid phosphatase accumulates in cells in which Srp54 synthesis has been repressed under the control of a regulated promoter, indicating that S. pombe SRP functions in protein targeting. In common with other Srp54 homologs, the S. pombe protein has a modular structure consisting of an amino-terminal G (GTPase) domain and a carboxyl-terminal M (methionine-rich) domain. We have analyzed the effects of 17 site-specific mutations designed to alter the function of each of the four GTPase consensus motifs individually. Several alleles, including some with relatively conservative amino acid substitutions, confer lethal or conditional phenotypes, indicating that GTP binding and hydrolysis are critical to the in vivo role of the protein. Two mutations (R to L at position 194 [R194L] and R194H) which were designed, by analogy to oncogenic mutations in rats, to dramatically decrease the catalytic rate and one (T248N) predicted to alter nucleotide binding specificity produce proteins that are unable to support growth at 18 degrees C. Consistent with its design, the R194L mutant hydrolyzes GTP at a reduced rate relative to wild-type Srp54 in enzymatic assays on immunoprecipitated proteins. In strains that also contain wild-type srp54, this mutant protein, as well as others designed to be locked in a GTP-bound conformation, exhibits temperature-dependent dominant inhibitory effects on growth, while a mutant predicted to be GDP locked does not interfere with the function of the wild-type protein. These results form the basis of a simple model for the role of GTP hydrolysis by Srp54 during the SRP cycle.

Amino Acid Sequence↗

PubMed1997

Identification of a minimal Alu RNA folding domain that specifically binds SRP9/14.

We have identified functionally and analyzed a minimal Alu RNA folding domain that is recognized by SRPphi14-9. Recombinant SRPphi14-9 is a fusion protein containing on a single polypeptide chain the sequences of both the SRP14 and SRP9 proteins that are part of the Alu domain of the signal recognition particle (SRP). SRPphi14-9 has been shown to bind to the 7SL RNA of SRP and it confers elongation arrest activity to reconstituted SRP in vitro. Alu RNA variants with homogeneous 3' ends were produced in vitro using ribozyme technology and tested for specific SRPphi14-9 binding in a quantitative equilibrium competition assay. This enabled identification of an Alu RNA of 86 nt (SA86) that competes efficiently with 7SL RNA for SRPphi14-9 binding, whereas smaller RNAs did not. The secondary structure of SA86 includes two stem-loops that are connected by a highly conserved bulge and, in addition, a part of the central adaptor stem that contains the sequence at the very 3' end of 7SL RNA. Circularly permuted variants of SA86 competed only if the 5' and 3' ends were joined with an extended linker of four nucleotides. SA86 can thus be defined as an autonomous RNA folding unit that does not require its 5' and 3' ends for folding or for specific recognition by SRPphi14-9. These results suggest that Alu RNA identity is determined by a characteristic tertiary structure, which might consist of two flexibly linked domains.

Animals↗

PubMed2005

Novel classification of idiopathic inflammatory myopathies based on overlap syndrome features and autoantibodies: analysis of 100 French Canadian patients.

Our objective was to improve the currently imperfect classifications of idiopathic inflammatory myopathies (IIM). In clinical practice, overlap features are common in IIM. This provided a rationale for positioning overlap clinical features at the core of a new classification system. We conducted a longitudinal study of 100 consecutive adult French Canadian patients with IIM. Clinical and laboratory data were obtained by retrospective chart review. Sera were analyzed for autoantibodies (aAbs) by protein A-assisted immunoprecipitation and double immunodiffusion. Overlap aAbs encompassed aAbs to synthetases, systemic sclerosis-associated aAbs, anti-signal recognition particle (SRP) and anti-nucleoporins. Patients were classified both at IIM diagnosis, based on data at presentation, and at the end of follow-up, based on cumulative findings. Three classifications were used: 1) the Bohan and Peter original classification, 2) a new version of that classification as modified by us, and 3) a novel clinicoserologic classification. As investigators were blinded to aAb results, the modified classification is strictly a clinical classification. Its core concept is the attribution of diagnostic significance to the presence of overlap features, that is, their presence resulted in a diagnosis of overlap myositis (OM). This approach allowed direct comparison with the original Bohan and Peter classification. By integrating aAb results to the modified classification, we also defined the clinicoserologic classification, which allowed to examine the added value of aAbs to diagnostic, therapeutic and prognostic stratification. Whereas polymyositis (PM) was the most common IIM according to the original classification, accounting for 45% of the cohort at diagnosis, its frequency fell to 14% with the modified classification. Conversely, while the frequency of myositis associated with connective tissue disease was 24% according to the original classification, the frequency of OM was 60% when using the modified classification. At last follow-up, the frequency of PM fell further to only 9%, while the frequency of OM rose to 67%. Systemic sclerosis was the most common connective tissue disease associated with IIM, accounting for 42.6% of OM patients and 29% of the cohort. The frequencies of overlap aAbs in the cohort and in OM patients were 48% and 70.5% (n =48/68), respectively. The presence of overlap aAbs at IIM diagnosis identified additional OM patients unrecognized by the modified classification. The sensitivity of the modified classification for OM at diagnosis was 87%, suggesting that clinicians may rely on the modified classification for identification of most OM patients, while awaiting results of aAb assays. The new classifications predicted the response to prednisone and IIM course. Using stringent definitions, IIM was classified as responsive or refractory after an adequate initial corticosteroid therapy, and the disease course as monophasic or chronic after a single adequate trial of prednisone. PM was always chronic and was associated with the highest rate (50%) of refractoriness to initial corticosteroid treatment. Dermatomyositis was almost always chronic (92% rate); however, its responsiveness to initial corticosteroid treatment was high (87%). OM was almost always responsive to corticosteroids (89%-100% rates). When OM patients were divided according to aAb subsets, anti-synthetase, SRP, or nucleoporin aAbs were markers for chronic myositis, whereas aAbs to U1RNP, Pm-Scl, or Ku were markers for monophasic myositis. We conclude that the original Bohan and Peter classification should be abandoned as it leads to misclassification of patients. Much of IIM is composed of OM. The proposed modified and clinicoserologic classifications have diagnostic, prognostic, and therapeutic value.

Adolescent↗

PubMed1990

In vitro biosynthesis and membrane translocation of the serine rich protein of Plasmodium falciparum.

The serine-rich protein (SERP) of Plasmodium falciparum is found within the parasitophorous vacuole. Exons 1 and 2 of the SERP gene were combined to a continuous open reading frame and expressed in a cell free translation/translocation system to study translocation of the protein across membranes. The protein was found to be translocated co-translationally across canine pancreatic microsomes. This process required the presence of the signal recognition particle, and it was accompanied by cleavage of a signal peptide. We conclude that the authentic SERP is exported from the parasite cell via the endoplasmic reticulum.

Animals↗

PubMed1988

Sequences beyond the cleavage site influence signal peptide function.

The earliest events in protein secretion include targeting to and translocation across the endoplasmic reticulum membrane. To dissect the mechanism by which signal sequences mediate translocation in eukaryotes, we are examining the behavior of fusion proteins and deletion mutants in cell-free systems. We demonstrate that the protein domain being translocated can have profound impact on the efficiency of the translocation process. Specifically, deletions in the mature prolactin "passenger" domain, beyond the signal cleavage site, reduce the efficiency of signal function. The effect of these deletions on signal function is observed when this signal sequence is in its normal position, at the amino terminus, and when internalized by the addition of 117 amino acids of chimpanzee alpha-globin. Alterations in the interaction of the deletion mutants with the signal recognition particle and with another component of the translocation system, signal peptidase, were observed. Our results suggest that subtle changes in sequences beyond the signal cleavage site can alter the efficiency of co-translational translocation by affecting various signal-receptor interactions.

Amino Acid Sequence↗

PubMed1988

Membrane orientation of rat gp110 as studied by in vitro translation.

We have recently isolated a full-length cDNA clone for a membrane glycoprotein with apparent Mr of 110,000, denoted gp110, that is expressed in rat liver, kidney, small intestine, and lung. Analysis of the amino acid sequence derived from cDNA sequencing demonstrated that there is a highly hydrophobic domain at the amino terminus (amino acid residues 1-29) that looks like the amino-terminal signal sequence. Using in vitro transcription and translation systems, we have expressed the full-length gp110 cDNA transcript, as well as transcripts derived from truncated gp110 cDNA that terminate translation at different sites. As expected, the amino-terminal signal sequence can promote the translocation across microsomal membranes of the downstream sequence. Like most translocations, the cotranslational translocation of the downstream sequence initiated by the gp110 amino-terminal signal sequence is mediated by signal recognition particle and docking protein. Unlike most amino-terminal signal sequences of lysosomal, secretory, and membrane proteins, the amino-terminal signal sequence of gp110 is not cleaved and may be involved in anchoring the polypeptide to the membrane. We, therefore, predict that the membrane orientation of gp110 is of type II with an extremely small amino-terminal cytoplasmic domain (6 residues).

Amino Acid Sequence↗

PubMed1987

Deletion analysis of the internal signal-anchor domain of the human asialoglycoprotein receptor H1.

The human asialoglycoprotein receptor H1 is a single-spanning membrane protein with the amino terminus facing the cytoplasm and the carboxy terminus exposed on the exoplasmic side of the plasma membrane. It has been shown earlier that the transmembrane segment, residues 38-65, functions as an internal signal directing protein synthesis to the endoplasmic reticulum and initiating membrane insertion. This process is co-translational and mediated by signal recognition particle (SRP). To identify subsegments within this region containing the signal information, we prepared deletion mutants at the level of the cDNA and analysed them in a wheat germ in vitro translation system with microsomes as the target membrane. Insertion and membrane anchoring were judged by the glycosylation of the protein, its resistance to exogenous protease and the extent to which it can be extracted from the microsomes by alkaline treatment. It was found that very small deletions already reduce the stability of membrane anchoring. However, nearly half of the transmembrane domain can be deleted, both from the amino-terminal and from the carboxy-terminal side, without completely abolishing membrane insertion. Several mutants, although not inserted, still interact with SRP. The results support the notion that the main feature of a signal sequence is a hydrophobic stretch of sufficient length (10-12 residues in our sequence), and indicate that recognition by SRP is not sufficient for membrane insertion.

Amino Acid Sequence↗

PubMed1995

Inefficient membrane targeting, translocation, and proteolytic processing by signal peptidase of a mutant preproparathyroid hormone protein.

A preproparathyroid hormone allele from a patient with familial isolated hypoparathyroidism was shown to have a single point mutation in the hydrophobic core of the signal sequence. This mutation, changing a cysteine to an arginine codon at the -8 position of the signal peptide, was associated with deleterious effects on the processing of preproparathyroid hormone to proparathyroid hormone in vitro. To examine the biochemical consequence(s) of this mutation, proteins produced by cell-free translation of wild-type and mutant cRNAs were used in assays that reconstitute the early steps of the secretory pathway. We find that the mutation impairs interaction of the nascent protein with signal recognition particle and the translocation machinery. Moreover, cleavage of the mutant signal sequence by solubilized signal peptidase is ineffective. The consequence of this mutation on processing and secretion of parathyroid hormone is confirmed in intact cells by pulse-chase experiments following transient expression of the mutant protein in COS-7 cells. The inability of the mutant signal sequence, however, to interfere with the targeting and processing of other secreted proteins does not support obstruction of the translocation apparatus as the mechanism underlying the dominant mode of inheritance of hypoparathyroidism in this family.

Alleles↗

PubMed1992

Functional expression of bovine 17 alpha-hydroxylase in COS 1 cells is dependent upon the presence of an amino-terminal signal anchor sequence.

Microsomal forms of eukaryotic cytochrome P450 proteins are integral membrane proteins of the endoplasmic reticulum (ER) membrane which are targeted to the ER via the signal recognition particle pathway. A hydrophobic amino terminus serves as a combined signal sequence and major membrane anchor (signal-anchor sequence) for the microsomal P450s. We have examined the insertion of bovine 17 alpha-hydroxylase (P45017 alpha) into the ER of COS 1 cells in order to evaluate the role of membrane insertion of the amino-terminal signal-anchor of microsomal P450s as a functional determinant for these enzymes. Previously, we have shown that deletion of the hydrophobic amino terminus from P45017 alpha reduced membrane targeting and insertion by 5-fold compared with the wild-type protein, abolished enzymatic activity, and resulted in an aberrant CO difference spectrum. In the present study we have replaced the amino terminus of P45017 alpha with two heterologous signal-anchor sequences, one that is similar and one that is very different from the P45017 alpha sequence. The chimeric proteins were expressed in COS 1 cells. Immunoblot analysis of isolated microsomal membranes show that the heterologous signal-anchor sequences functioned to target the P45017 alpha protein to the ER. Enzymatic assays in intact COS 1 cells indicate that both the chimeric proteins are efficient 17 alpha-hydroxylase enzymes. The amino terminus of P45017 alpha was also replaced with a sequence that is not a signal-anchor, and the expressed protein was neither targeted to the ER nor was functional in COS 1 cells. In conclusion, both the structure and catalytic activity of P45017 alpha in COS 1 cells is dependent upon an amino-terminal sequence that functions as a signal-anchor sequence and not upon the precise sequence of the amino terminus.

Amino Acid Sequence↗

PubMed1987

A short amino-terminal segment of microsomal cytochrome P-450 functions both as an insertion signal and as a stop-transfer sequence.

Co-translational insertion of liver microsomal cytochrome P-450 into the endoplasmic reticulum membrane is mediated by the signal recognition particle (SRP) and the presence in the cytochrome molecule of a signal sequence that can be recognized by SRP has been postulated. To locate this signal sequence, six hybrid cDNAs were constructed in which various segments of a cDNA for a rabbit liver cytochrome P-450 are fused with a cDNA or its fragment encoding yeast porin (an outer mitochondrial membrane protein) or with a cDNA for pre-interleukin 2 (a secretory protein) from which the 5'-terminal portion encoding most of its signal sequence had been removed. These hybrid cDNAs were inserted into an SP-6 transcription vector and transcribed in vitro. The mRNAs thus synthesized were translated in a cell-free system in the presence of rough microsomes. It was thus found that only those chimeric proteins containing (at their amino-terminal end) the amino-terminal cytochrome P-450 segments consisting of greater than or equal to 29 amino acid residues were co-translationally inserted into the membrane in an SRP-dependent fashion. These proteins were, however, neither processed nor translocated across the membrane. These findings, coupled with the observation that the major portion of these proteins, when inserted into the membrane, was degraded by trypsin, led to the conclusion that a short amino-terminal segment (less than 29 residues) of the cytochrome P-450 functions not only as an insertion signal but also as a stop-transfer sequence. This segment is, therefore, similar to the internal signal of type II plasma membrane proteins, but differs from the latter in the topogenic function.

Amino Acid Sequence↗

PubMed1991

Ribosome binding to the endoplasmic reticulum: a 180-kD protein identified by crosslinking to membrane-bound ribosomes is not required for ribosome binding activity.

We have used the membrane-impermeable, thiol-cleavable, crosslinker 3,3'-dithio bis (sulfosuccinimidylpropionate) to identify proteins that are in the vicinity of membrane-bound ribosomes of the RER. A specific subset of RER proteins was reproducibly crosslinked to the ribosome. Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35-kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent. The most prominent polypeptide among the crosslinked products was a 180-kD protein that has recently been proposed to be a ribosome receptor (Savitz, A.J., and D.I. Meyer, 1990. Nature (Lond.). 346: 540-544). RER membrane proteins were reconstituted into liposomes and assayed with radiolabeled ribosomes to determine whether ribosome binding activity could be ascribed to the 180-kD protein. Differential detergent extraction was used to prepare soluble extracts of microsomal membrane vesicles that either contained or lacked the 180-kD protein. Liposomes reconstituted from both extracts bound ribosomes with essentially identical affinity. Additional fractionation experiments demonstrated that the bulk of the ribosome binding activity present in detergent extracts of microsomal membranes could be readily resolved from the 180-kD protein by size exclusion chromatography. Taken together, we conclude that the 180-kD protein is in the vicinity of membrane bound ribosomes, yet does not correspond to the ribosome receptor.

Animals↗

PubMed1988

7SL RNA from Schizosaccharomyces pombe is encoded by a single copy essential gene.

We have identified an abundant ribonucleoprotein particle from Schizosaccharomyces pombe with properties related to those of the vertebrate signal recognition particle (SRP), including cytoplasmic localization, association with microsomes and ribosomes at low, but not high, salt concentrations and high resistance to micrococcal nuclease. The 256-nucleotide RNA component carries a 5'-triphosphate group and shows close secondary structure, and limited primary sequence homology to vertebrate 7SL RNA. 7SL-like RNAs were also detected in a number of other fungi. The single copy gene (SRP7) encoding S.pombe 7SL was disrupted by insertion of a transposon carrying the selective marker LEU2, and the disrupted gene was used to replace one chromosomal SRP7 gene in a diploid strain. Haploid srp7[unk] strains fail to germinate.

Journal Article↗

PubMedDate not supplied

Xenopus oocytes can secrete bacterial beta-lactamase.

Most secretory proteins are synthesized as precursor polypeptides carrying N-terminal, hydrophobic sequences which, by means of a signal recognition particle (SRP), trigger the membrane transfer of the polypeptide and are subsequently cleaved off. The signal sequences appear to be interchangeable between prokaryotes and eukaryotes. In bacteria, secretion only involves the crossing of a membrane, whereas in eukaryotes the secretory process can be separated into two distinct phases: translocation across the membrane of the rough endoplasmic reticulum and subsequent intraluminal transport by processes involving vesicle budding and fusion. Since secretory proteins must be distinguished from other soluble proteins destined for various sites in the reticular system, it is conceivable that eukaryotic secretory proteins possess additional markers distinct from the signal peptide to guide the polypeptide after its transfer through the membrane. Proteins are secreted at different rates from a eukaryotic cell, suggesting a role in intracellular transport for receptors with differing affinities for some topogenic features in secretory proteins. We have tested this possibility by introducing into the lumen of eukaryotic rough endoplasmic reticulum a prokaryotic protein which, by virtue of its origin, had not been adapted to the eukaryotic secretory pathway. We reasoned that secretion of the bacterial protein would indicate that after membrane transfer no topogenic signal(s) and corresponding recognition system(s) are required. We report here that this is indeed the case.

Animals↗

PubMed1999

Functional changes in the structure of the SRP GTPase on binding GDP and Mg2+GDP.

Ffh is a component of a bacterial ribonucleoprotein complex homologous to the signal recognition particle (SRP) of eukaryotes. It comprises three domains that mediate both binding to the hydrophobic signal sequence of the nascent polypeptide and the GTP-dependent interaction of Ffh with a structurally homologous GTPase of the SRP receptor. The X-ray structures of the two-domain 'NG' GTPase of Ffh in complex with Mg2+GDP and GDP have been determined at 2.0 A resolution. The structures explain the low nucleotide affinity of Ffh and locate two regions of structural mobility at opposite sides of the nucleotide-binding site. One of these regions includes highly conserved sequence motifs that presumably contribute to the structural trigger signaling the GTP-bound state. The other includes the highly conserved interface between the N and G domains, and supports the hypothesis that the N domain regulates or signals the nucleotide occupancy of the G domain.

Amino Acid Sequence↗