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PubMed2000

Oligomerization of pro-opiomelanocortin is independent of pH, calcium and the sorting signal for the regulated secretory pathway.

Studies indicate that pro-opiomelanocortin (POMC) is sorted to the regulated secretory pathway by binding to a sorting receptor identified as membrane-bound carboxypeptidase E (CPE) [Cool et al. (1997) Cell 88, 73-83]. The efficiency of this sorting mechanism could be enhanced if POMC molecules were to self-associate to form oligomers, prior or subsequent to binding to CPE. Using cross-linking and gel filtration techniques, we demonstrated that POMC forms oligomers at both neutral and acidic pHs and calcium was not necessary. delta N-POMC, which lacks the N-terminal sorting signal for the regulated secretory pathway, also formed similar oligomers, indicating that the sorting and oligomerization domains are different.

Animals↗
PubMed2000

Large-scale predictions of secretory proteins from mammalian genomic and EST sequences.

Machine learning techniques have improved predictions of secretory proteins from protein, genomic and expressed sequence tag (EST) sequences. Artificial neural networks, physical sequence analysis using high-performance optimization, and hidden Markov models identify extremely variable signal peptides (the vehicles of protein transport across the endoplasmic reticulum membrane), transmembrane segments, and specific extracellular and intracellular domains as indicators of possible roles in the intercellular and intracellular chemical signaling pathways. The major role of peptide hormones, blood coagulation factors, carcinogenesis agents, and other secretory proteins in orchestrating multicellular life indicates pharmacological potential in the cure of major diseases and numerous biotechnological applications.

Animals↗
PubMed1997

Characterization of a novel tyrosine phosphorylated 100-kDa protein that binds to SHP-2 and phosphatidylinositol 3'-kinase in myeloid cells.

Fms is a tyrosine kinase-containing receptor for macrophage colony-stimulating factor (M-CSF) that regulates survival, growth, and differentiation of cells along the monocyte/macrophage lineage. M-CSF stimulation of murine myeloid FDC-P1 cells expressing Fms resulted in the tyrosine phosphorylation of a number of signal transduction proteins, including an unidentified 100-kDa protein. This 100-kDa protein associated with the tyrosine phosphatase SHP-2 but not with the related phosphatase SHP-1. The kinetics of tyrosine phosphorylation of p100 and SHP-2 suggest that p100 may be a direct substrate of SHP-2. p100 bound directly to the SH2 domains of both SHP-2 and the p85 subunit of phosphatidylinositol 3'-kinase. The 100-kDa protein did not appear to bind directly to Fms, Ship, Cbl, Shc, or Grb2, although all of these proteins were coimmunoprecipitated with p85 after M-CSF stimulation. Association of p100 with SHP-2 and p85 did not require the major autophosphorylation sites on Fms nor binding of p85 to Fms. A tyrosine phosphorylated protein of 100 kDa also coprecipitated with SHP-2 from several other myeloid cell lines after M-CSF stimulation but was not seen in immunoprecipitates from Rat2 fibroblasts expressing Fms. Stimulation of FDC-P1 cells with additional cytokines also resulted in coprecipitation of a 100-kDa protein with SHP-2. p100 may therefore be a common component of the signaling pathways of cytokine receptors in myeloid cells.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Secretion and circular dichroism analysis of the C-terminal signal peptides of HlyA and LktA.

The secretion of the 107 kDa hemolysin A (HlyA) from Escherichia coli is mediated by membrane proteins hemolysin B (HlyB) and hemolysin D (HlyD). The signal for transport has been mapped to the C-terminal 60 amino acids of the HylA molecule. We have shown previously that the C-terminal 70 amino acids of leukotoxin (LktA) from Pasteurella hemolytica can substitute functionally for the HlyA signal sequence. This 70 amino acid peptide contains little primary sequence similarity to the HlyA signal sequence, and we have hypothesized that these signal sequences assume a similar higher-order structure which is recognized by the HlyB/D transporter. In the present study, we have expressed and purified small peptides containing the C-terminal 61 amino acids of HlyA and the C-terminal 70 amino acids of LktA. We show that these signal peptides are sufficient for secretion from E. coli in a HlyB/D dependent manner. Circular dichroism analyses show that both molecules exhibit common biophysical properties. In aqueous solution, they appear to be mainly unstructured, but in a membrane mimetic environment they assume a helical secondary structure. The conformational change observed for both peptides going from an aqueous to a membrane mimetic environment may be an important feature of these signal sequences necessary for their recognition and transport.

Bacterial Proteins↗
PubMed1994

Identification of the nucleolar targeting signal of human angiogenin.

Angiogenin is endocytosed by subconfluent endothelial cells, translocated to the nucleus and accumulates in the nucleolus. It also localizes into the nucleolus of digitonin-permeabilized endothelial cells. The peptide RRRGL corresponding to residues 31-35 of human angiogenin specifically targets non-nuclear carrier proteins such as albumin, an anti-human nucleolus monoclonal antibody and R33A angiogenin to the nucleolus of permeabilized endothelial cells. Proteins conjugated with a "mutant" peptide, RRAGL, are not imported. Fluorescein isothiocyanate-conjugated RRRGL is also rapidly imported into the nucleus and localized to the nucleolus, whereas the "mutant" peptide is not. Residue R33 is essential for nuclear translocation and R31 and R32 appear to modulate this process. Thus, 31RRRGL35 is a nuclear localization signal responsible for the nucleolar targeting of human angiogenin.

Amino Acid Sequence↗
PubMed2000

Requirement for the COOH-terminal pro-sequence in the translocation of aqualysin I across the cytoplasmic membrane in Escherichia coli.

Aqualysin I from Thermus aquaticus YT-1 is an extracellular subtilisin-type serine protease. The protease is synthesized as a distinct precursor composed of four functional domains: an N-terminal signal sequence, an N-terminal pro-sequence, a protease domain, and a C-terminal pro-sequence. The N-terminal pro-sequence is essential for the production of active aqualysin I while the C-terminal pro-sequence is required for extracellular secretion of aqualysin I. In an E. coli expression system, the function of C-terminal pro-sequence in the translocation of aqualysin I across the cytoplasmic membrane was investigated. More than 60-70% of the total activity was detected in the cytoplasmic fraction in the deletion mutations of the C-terminal pro-sequence while less than 30% was found in this fraction in wild type. In addition, in vitro processing of aqualysin I precursors with these mutations to a mature form promptly occurred and the folding into active aqualysin I was rapid. These results suggest that the C-terminal pro-sequence, probably in conjunction with the signal sequence, facilitates the translocation of the precursor across the cytoplasmic membrane by preventing the precursor from taking on an active conformation.

Cell Membrane↗
PubMed2000

Aberrant signal peptide cleavage of collagen X in Schmid metaphyseal chondrodysplasia. Implications for the molecular basis of the disease.

Schmid metaphyseal chondrodysplasia results from mutations in the collagen X (COL10A1) gene. With the exception of two cases, the known mutations are clustered in the C-terminal nonhelical (NC1) domain of the collagen X. In vitro and cell culture studies have shown that the NC1 mutations result in impaired collagen X trimer assembly and secretion. In the two other cases, missense mutations that alter Gly(18) at the -1 position of the putative signal peptide cleavage site were identified (Ikegawa, S., Nakamura, K., Nagano, A., Haga, N., and Nakamura, Y. (1997) Hum. Mutat. 9, 131-135). To study their impact on collagen X biosynthesis using in vitro cell-free translation in the presence of microsomes, and cell transfection assays, these two mutations were created in COL10A1 by site-directed mutagenesis. The data suggest that translocation of the mutant pre-alpha1(X) chains into the microsomes is not affected, but cleavage of the signal peptide is inhibited, and the mutant chains remain anchored to the membrane of microsomes. Cell-free translation and transfection studies in cells showed that the mutant chains associate into trimers but cannot form a triple helix. The combined effect of both the lack of signal peptide cleavage and helical configuration is impaired secretion. Thus, despite the different nature of the NC1 and signal peptide mutations in collagen X, both result in impaired collagen X secretion, probably followed by intracellular retention and degradation of mutant chains, and causing the Schmid metaphyseal chondrodysplasia phenotype.

Animals↗
PubMed1997

Spectroscopic studies of the C-terminal secretion signal of the Serratia marcescens haem acquisition protein (HasA) in various membrane-mimetic environments.

The structure of a peptide comprising the last 56 C-terminal residues of the Serratia marcescens haem acquisition protein (HasA) secreted by an ATP-binding cassette exporter was examined by 1H-NMR, circular dichroic and fluorescence spectroscopies. The peptide, which contains the secretion signal of HasA, is efficiently secreted by the HasA transporter. It is largely unstructured and flexible in aqueous buffer solution, but its helical content increases upon addition of trifluoroethanol, detergents and lipids. By circular dichroism, a stable helical conformation is observed between 20% and 70% (by vol.) trifluoroethanol. The 1H-NMR spectrum was analysed at these two trifluoroethanol concentrations; residues 7-15, 21-30 and 40-50 were shown to form relatively stable helices. In the presence of neutral detergent, alpha-helix is induced to a similar extent upon micelle formation; in this case, fluorescence data indicate that at least the N-terminus of the peptide interacts with the micelle. In the presence of negatively charged detergent, alpha-helix is induced before micelle formation and the N-terminus of the peptide seems not to be involved in this interaction. In the presence of negatively charged liposomes, the peptide interacts with the vesicle, again inducing a helical conformation. However, the helical content remains lower than upon addition of trifluoroethanol or neutral micelles. These results are compared to those previously obtained with the secretion signal of one of the Erwinia chrysanthemi metalloproteases which are transported efficiently by the HasA transporter. Both signals exhibit similar conformational features, despite their low sequence similarity.

ATP-Binding Cassette Transporters↗
PubMed2002

Systematic site-directed mutagenesis of human protein SRP54: interactions with signal recognition particle RNA and modes of signal peptide recognition.

The amino acid residues of human protein SRP54 which are required for binding to SRP RNA were identified by generating 40 nonoverlapping tri-alanine alterations within its methionine-rich M-domain (SRP54M). The mutant polypeptides were expressed in Escherichia coli, and their ability to bind to human and Methanococcus jannaschii SRP RNA were determined in vitro. Residues at positions 379-387, 394-396, 400-405, and 409-411 of human SRP54 were within the predicted RNA binding site, and their alteration abolished the binding activities of the mutant polypeptides as expected. Changes at positions 418-423 had intermediate effects. Polypeptides containing mutations of 328-TLR-330 were inactive although these residues were far away from the presumed RNA binding site in the crystal structure of the free protein. Using the structures of the E. coli Ffh/4.5S core and of the human SRP54m dimer as templates, a molecular model of the complex between human SRP RNA helix 8 and a single SRP54M molecule was constructed in which Leucine 329 was positioned in closer proximity to the RNA binding domain. This representation was supported by studies of the SRP54m monomer/dimer ratio using gel filtration. The results were consistent with a change in the shape of the signal peptide binding groove upon binding of SRP54 to SRP RNA. We propose that the SRP RNA and a small region centered at a bulky nonpolar amino acid residue at position 329 of protein SRP54 play a critical role in the SRP-dependent binding and release of signal peptides.

Amino Acid Sequence↗
PubMed2003

Role of a conserved amino-terminal sequence in the ecotropic MLV receptor mCAT1.

The amino-terminus of mCAT1 and homologous proteins is predicted to form a positively charged, amphipathic alpha helix on the cytoplasmic side of the plasma membrane. Peptides with similar sequence motifs often provide membrane anchors, protein-protein interaction domains, or intracellular transport-targeting signals. Deleting most of the cytoplasmic N-terminal sequence of mCAT1 led to reduced expression on the cell surface and accumulation in the endoplasmic reticulum but did not abrogate receptor function. Surprisingly, when the N-terminal 36 or 18 amino acids of mCAT1 were fused to green fluorescent protein (gfp), gfp accumulated almost exclusively in mitochondria. Mitochondrial targeting depended on arginines at positions 15 and 16 and was inhibitable by downstream transmembrane sequences. Although the full-length mCAT1 was not detected in mitochondria, the mitochondrial-targeting property of the N-terminal sequence fused to gfp is conserved in orthologous and paralogous proteins that diverged approximately 80 million years ago, suggesting a conserved biological function. We propose that the conserved N-terminal motif of CAT proteins provides a regulatable signal for transport to, or retention in, different cell membrane compartments.

Amino Acid Motifs↗
PubMed2002

Distinct modes of signal recognition particle interaction with the ribosome.

Signal recognition particle (SRP), together with its receptor (SR), mediates the targeting of ribosome-nascent chain complexes to the endoplasmic reticulum. Using protein cross-linking, we detected distinct modes in the binding of SRP to the ribosome. During signal peptide recognition, SRP54 is positioned at the exit site close to ribosomal proteins L23a and L35. When SRP54 contacts SR, SRP54 is rearranged such that it is no longer close to L23a. This repositioning may allow the translocon to dock with the ribosome, leading to insertion of the signal peptide into the translocation channel.

Animals↗
PubMed1995

Physical and conformational properties of synthetic idealized signal sequences parallel their biological function.

Transported proteins often contain an extension sequence called the signal peptide. The alkaline phosphatase (PhoA) signal sequence represents a typical signal peptide for comparison to idealized sequences both in vivo and in vitro. We have designed a series of idealized signal sequences which vary in amino terminal charge and core region hydrophobicity with minimal variation in amino acid composition. The idealized core regions contain different proportions of leucine and alanine residues, effectively producing hydrophobicities above and below the threshold level required for efficient secretion. The flanking amino and carboxyl termini were designed to maintain the general features and relative hydrophobicity of their counterparts in the wild-type PhoA signal sequence. Using the phoA gene, the signal peptide region was modified to generate mutants corresponding to the model sequences. Transport studies in Escherichia coli confirmed that completely idealized signal sequences, which lack a helix-breaking proline or glycine residue, can be functional if the core region is sufficiently hydrophobic and that one positively charged residue in the amino terminus is adequate for efficient transport. The corresponding peptides were chemically synthesized and exhibited HPLC retention times that reflect the relative hydrophobicities of the sequences. Structural analyses of the isolated peptides by circular dichroism demonstrate solvent dependence and exceptionally stable alpha-helix formation by the functional signal peptides in trifluoroethanol. Although leucine and alanine residues are often predicted to have similar propensities for forming an alpha-helix, considerably higher alpha-helical content is observed in the signal peptides which contain predominantly polyleucine core regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed2002

Dependence of prePhoA-phospholipid interaction in vivo and in vitro on charge of signal peptide N-terminus and content of anionic phospholipids in membranes.

Replacement of the positively charged signal peptide with neutral or negatively charged peptides due to substitution of Lys(-20) in the N-terminal region of the signal peptide leads to decreases in the rate of prePhoA membrane translocation in vivo and in the efficiency of prePhoA insertion into liposomes in vitro. The effect of anionic phospholipids on prePhoA insertion into model membranes is determined by the signal peptide N-terminus charge, while the dependence of prePhoA translocation across the cytoplasmic membrane in vivo is not, under the studied variations in the content of anionic phospholipids. This is evidence of the possibility of direct electrostatic interaction between the signal peptide N-terminus and anionic phospholipids, which in vivo, however, seems to involve some proteins of the Sec machinery.

Anions↗
PubMed2000

Polar transmembrane domains target proteins to the interior of the yeast vacuole.

Membrane proteins transported to the yeast vacuole can have two fates. Some reach the outer vacuolar membrane, whereas others enter internal vesicles, which form in late endosomes, and are ultimately degraded. The vacuolar SNAREs Nyv1p and Vam3p avoid this fate by using the AP-3-dependent pathway, which bypasses late endosomes, but the endosomal SNARE Pep12p must avoid it more directly. Deletion analysis revealed no cytoplasmic sequences necessary to prevent the internalization of Pep12p in endosomes. However, introduction of acidic residues into the cytoplasmic half of the transmembrane domain created a dominant internalization signal. In other contexts, this same feature diverted proteins from the Golgi to endosomes and slowed their exit from the endoplasmic reticulum. The more modestly polar transmembrane domains of Sec12p and Ufe1p, which normally serve to hold these proteins in the endoplasmic reticulum, also cause Pep12p to be internalized, as does that of the vacuolar protein Cps1p. It seems that quality control mechanisms recognize polar transmembrane domains at multiple points in the secretory and endocytic pathways and in endosomes sort proteins for subsequent destruction in the vacuole. These mechanisms may minimize the damaging effects of abnormally exposed polar residues while being exploited for the localization of some normal proteins.

Amino Acid Sequence↗
PubMed2003

The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway.

The Escherichia coli cytoplasmic protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding protein (MBP). Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway. When DsbAss is fused to MBP, MBP also is directed to the SRP pathway. We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized. However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA. These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway. Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, along with gene fusion approaches, a sensitive assay system for signal sequences that utilize the SRP pathway.

Adenosine Triphosphatases↗
PubMed1999

Effect of transforming RNA on the synthesis of a protein with a secretory signal sequence in vitro.

U5 small nuclear RNA itself can act as a clastogenic and transforming agent when transfected into cells. In the previous work, the 3' half of the U5 small nuclear RNA first stem structure (designated RNA3S) was capable of driving normal cells into tumorigenic cells when expressed with a poly(A) tail (RNA3S+). This transformation critically depended upon the polypurine sequence GGAGAGGAA in RNA3S+. In this work, we first examined the pre-beta-lactamase and luciferase (model secretory and nonsecretory proteins) translation with the in vitro synthesized RNA3S in rabbit reticulocyte lysate. The capped RNA3S with a poly(A) tail suppressed the translation. In addition, the polypurine sequence played a crucial role in affecting the secretory protein synthesis, indicating a primary action of RNA3S+. Further studies revealed that the oligodeoxynucleotides, corresponding to the polypurine and its antisense sequences, directly contacted 28 S rRNA in ribosome and 7SL RNA in signal recognition particle, respectively, and differentially affected the nascent chain elongation of secretory protein synthesis. These results suggest that RNA3S+ blocks a physiological regulatory function played by signal recognition particle and the ribosome in the secretory protein synthesis and support the idea that the transformation might result from a repressed cellular activity.

Animals↗
PubMed2000

Both transmembrane domains of SecG contribute to signal sequence recognition by the Escherichia coli protein export machinery.

A chimeric protein containing the uncleaved signal sequence of plasminogen activators inhibitor-2 (PAI2) fused to alkaline phosphatase (AP) interferes with Escherichia coli protein export and arrests growth. Suppressors of this toxicity include secG mutations that define the Thr-41-Leu-42-Phe-43 (TLF) domain of SecG. These mutations slow down the export of PAI2-AP. Another construct encoding a truncated PAI2 signal sequence (hB-AP) is also toxic. Most suppressors exert their effect on both chimeric proteins. We describe here five secG suppressors that only suppress the toxicity of hB-AP and selectively slow down its export. These mutations do not alter the TLF domain: three encode truncated SecG, whereas two introduce Arg residues in the transmembrane domains of SecG. The shortest truncated protein only contains 13 residues of SecG, suggesting that the mutation is equivalent to a null allele. Indeed, a secG disruption selectively suppresses the toxicity of hB-AP. However, the missense mutations are not null alleles. They allow SecG binding to SecYE, although with reduced affinity. Furthermore, these mutated SecG are functional, as they facilitate the export of endogenous proteins. Thus, SecG participates in signal sequence recognition, and both transmembrane domains of SecG contribute to ensure normal signal sequence recognition by the translocase.

Alkaline Phosphatase↗