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PubMed1997

A genetic selection for isolating cDNAs encoding secreted proteins.

We describe a simple, rapid technique for simultaneously isolating large numbers of cDNAs encoding secreted proteins. The technique makes use of a facile genetic selection performed in a strain of Saccharomyces cerevisiae deleted for its endogenous invertase gene. A cDNA cloning vector which carries a modified invertase gene lacking its leader sequence is used in conjunction with this strain. Heterologous secreted genes fused appropriately upstream of this defective invertase provide the necessary signals to restore secretion, allowing the yeast to grow on sugars such as sucrose or raffinose. This microbial growth selection facilitates scanning cDNA libraries containing millions of clones, enabling the wholesale identification of novel secreted proteins without the need for specific bioassays. The technique is similar to one previously described (Klein et al. (1996) Proc. Natl. Acad. Sci. USA 93, 7108-7113). We describe results using a cDNA library derived from activated human peripheral blood mononuclear cells (PBMC). Genes identified from this library encoded signal sequences of proteins of diverse structure, function, and cellular location such as cytokines, type 1 and type 2 transmembrane proteins, and proteins found in intracellular organelles. In addition, a number of novel secreted proteins were identified, including a chemokine and a novel G-protein-coupled receptor. Since signal sequences possess features conserved throughout evolution, the procedure can be used to isolate genes encoding secreted proteins from both eukaryotes and prokaryotes.

Amino Acid Sequence↗

PubMed2002

Dual-colour imaging of membrane protein targeting directed by poa semilatent virus movement protein TGBp3 in plant and mammalian cells.

The movement function of poa semilatent hordeivirus (PSLV) is mediated by the triple gene block (TGB) proteins, of which two, TGBp2 and TGBp3, are membrane proteins. TGBp3 is localized to peripheral bodies in the vicinity of the plasma membrane and is able to re-direct TGBp2 from the endoplasmic reticulum (ER) to the peripheral bodies. For imaging of TGBp3-mediated protein targeting, PSLV TGBp3 tagged with a red fluorescent protein (DsRed) was used. Coexpression of DsRed-TGBp3 with GFP targeted to the ER lumen (ER-GFP) demonstrated that ER-GFP was contained in typical ER structures and peripheral bodies formed by TGBp3 protein, suggesting an ER origin for these bodies. In transient coexpression with viral membrane proteins tagged with GFP, DsRed-TGBp3 directed to the peripheral bodies the homologous TGBp2 protein and two unrelated membrane proteins, the 6 kDa movement protein of beet yellows closterovirus and the putative movement protein encoded by the genome component 4 of faba bean necrotic yellows nanovirus. However, coexpression of TGBp3 with GFP derivatives targeted to the ER membranes by artificial hydrophobic tail sequences suggested that targeting to the ER membranes per se was not sufficient for TGBp3-directed protein trafficking to peripheral bodies. TGBp3-induced targeting of TGBp2 also occurred in mammalian cells, indicating the universal nature of the protein trafficking signals and the cotargeting mechanism.

Animals↗

PubMed2004

A structural step into the SRP cycle.

Co-translational targeting of secretory and membrane proteins to the translocation machinery is mediated by the signal recognition particle (SRP) and its membrane-bound receptor (SR) in all three domains of life. Although the overall composition of the SRP system differs, the central ribonucleoprotein core and the general mechanism of GTP-dependent targeting are highly conserved. Recently, structural studies have contributed significantly to our understanding of the molecular organization of SRP. SRP appears as a structurally flexible particle modulated and regulated by its interactions with the ribosome-nascent chain complex, the translocon and the SR. The SRP core (SRP54 with its cognate RNA binding site) plays a central role in these interactions and communicates the different binding states by long-range interdomain communication. Based on recent structures of SRP54, a model for signal peptide binding stimulating the GTP affinity during the first step of the SRP cycle is presented. The model is placed in the context of the recent structures of mammalian SRP bound to a ribosome-nascent chain complex and of a subcomplex of SRP-SR.

Guanosine Triphosphate↗

PubMed2004

Subcellular localization of adenine and xanthine phosphoribosyltransferases in Leishmania donovani.

The subcellular location of a protein is a critical factor in its physiological function and an important consideration in therapeutic paradigms that target the protein. Because Leishmania donovani cannot synthesize purine nucleotides de novo, they rely predominantly upon therapeutically germane phosphoribosyltransferase (PRT) enzymes, hypoxanthine-guanine PRT (HGPRT), adenine PRT (APRT), and xanthine PRT (XPRT), for purine acquisition from the host. Previous studies have shown that the L. donovani HGPRT is localized to the glycosome, a fuel-metabolizing microbody that is unique to kinetoplastid parasites [J. Biol. Chem. 273 (1998) 1534]. The sequences of the other two PRTs indicate that XPRT, but not APRT, possesses a COOH-terminal tripeptide that mediates protein targeting to the glycosome. To determine definitively the intracellular milieu of APRT and XPRT, polyclonal antibodies were raised to each recombinant protein. APRT and XPRT were then shown by immunofluorescence to be localized to the cytosol and glycosome, respectively. The glycosomal milieu for XPRT was also verified by immunoelectron microscopy. Amputation of the glycosomal targeting signal from XPRT resulted in protein mislocalization to the cytosol, but the cytosolic xprt was still functional with respect to purine salvage. These studies establish that APRT is cytosolic and XPRT, like the homologous HGPRT, is glycosomal and demonstrate that a mutant xprt protein that mislocalizes to the cytosol is still functional and supports parasite viability.

Adenine Phosphoribosyltransferase↗

PubMed2006

Secretory protein enrichment and analysis: an optimized approach applied on cancer cell lines using 2D LC-MS/MS.

Reliable methods for profiling secretory proteins are highly desirable for the identification of biomarkers of disease progression. Secreted proteins are often masked by high amounts of protein supplements in the culture medium. We have developed an efficient method for the enrichment and analysis of the secretome of different cancer cell lines, free of essential contaminants. The method is based on the optimization of cell incubation conditions in protein-free medium. Secreted proteins are concentrated and fractionated using a reversed-phase tC2 Sorbent, followed by peptide mass fingerprinting for protein identification. An average of 88 proteins were identified in each cancer cell line, of which more than 76% are known to be secreted, possess a signal peptide or a transmembrane domain. Given the importance of secreted proteins as a source for early detection and diagnosis of disease, this approach may help to discover novel candidate biomarkers with potential clinical significance.

Cell Culture Techniques↗

PubMed2004

A host-targeting signal in virulence proteins reveals a secretome in malarial infection.

Malaria parasites secrete proteins across the vacuolar membrane into the erythrocyte, inducing modifications linked to disease and parasite survival. We identified an 11-amino acid signal required for the secretion of proteins from the Plasmodium falciparum vacuole to the human erythrocyte. Bioinformatics predicted a secretome of >320 proteins and conservation of the signal across parasite species. Functional studies indicated the predictive value of the signal and its role in targeting virulence proteins to the erythrocyte and implicated its recognition by a receptor/transporter. Erythrocyte modification by the parasite may involve plasmodial heat shock proteins and be vastly more complex than hitherto realized.

Amino Acid Motifs↗

PubMed2006

Selective photoaffinity labeling identifies the signal peptide binding domain on SecA.

SecA, an ATPase crucial to the Sec-dependent translocation machinery in Escherichia coli, recognizes and directly binds the N-terminal signal peptide of an exported preprotein. This interaction plays a central role in the targeting and transport of preproteins via the SecYEG channel. Here we identify the signal peptide binding groove (SPBG) on SecA addressing a key issue regarding the SecA-preprotein interaction. We employ a synthetic signal peptide containing the photoreactive benzoylphenylalanine to efficiently and specifically label SecA containing a unique Factor Xa site. Comparison of the photolabeled fragment from the subsequent proteolysis of several SecAs, which vary only in the location of the Factor Xa site, reveals one 53 residue segment in common with the entire series. The covalently modified SecA segment produced is the same in aqueous solution and in lipid vesicles. This spans amino acid residues 269 to 322 of the E. coli protein, which is distinct from a previously proposed signal peptide binding site, and contributes to a hydrophobic peptide binding groove evident in molecular models of SecA.

Adenosine Triphosphatases↗

PubMed2005

Signal peptide hydrophobicity is critical for early stages in protein export by Bacillus subtilis.

Signal peptides that direct protein export in Bacillus subtilis are overall more hydrophobic than signal peptides in Escherichia coli. To study the importance of signal peptide hydrophobicity for protein export in both organisms, the alpha-amylase AmyQ was provided with leucine-rich (high hydrophobicity) or alanine-rich (low hydrophobicity) signal peptides. AmyQ export was most efficiently directed by the authentic signal peptide, both in E. coli and B. subtilis. The leucine-rich signal peptide directed AmyQ export less efficiently in both organisms, as judged from pulse-chase labelling experiments. Remarkably, the alanine-rich signal peptide was functional in protein translocation only in E. coli. Cross-linking of in vitro synthesized ribosome nascent chain complexes (RNCs) to cytoplasmic proteins showed that signal peptide hydrophobicity is a critical determinant for signal peptide binding to the Ffh component of the signal recognition particle (SRP) or to trigger factor, not only in E. coli, but also in B. subtilis. The results show that B. subtilis SRP can discriminate between signal peptides with relatively high hydrophobicities. Interestingly, the B. subtilis protein export machinery seems to be poorly adapted to handle alanine-rich signal peptides with a low hydrophobicity. Thus, signal peptide hydrophobicity appears to be more critical for the efficiency of early stages in protein export in B. subtilis than in E. coli.

Alanine↗

PubMed2005

YidC-dependent translocation of green fluorescence protein fused to the FliP cleavable signal peptide.

Escherichia coli FliP is a rare bacterial polytopic membrane protein synthesized with a cleavable, highly hydrophobic signal peptide. More hydrophilic Tat-dependent or Sec-dependent signal peptide is functionally capable of substituting for the FliP signal peptide, but a signal anchor of inner membrane protein fails to do so. To assess the intrinsic characteristics of the FliP signal peptide in mediating protein translocation, we fused it to green fluorescence protein and observed that the translocation of the chimera (FliPss-GFP) was dependent of Ffh, SecA, SecY and SecD. In addition, we showed for the first time the involvement of YidC in protein translocation across the inner membrane.

Escherichia coli↗

PubMed1997

Orientation in lipid bilayers of a synthetic peptide representing the C-terminus of the A1 domain of shiga toxin. A polarized ATR-FTIR study.

The interaction of a synthetic peptide representing the C-terminal 27 amino acids of the A1 domain of Shiga toxin (residues 220-246) with acidic phospholipid model membranes was characterized by FTIR spectroscopy. This peptide resembles a signal sequence and may mediate the translocation of the catalytic A1 chain of Shiga toxin to the cytoplasm following its retrograde transport to the lumenal compartment of the endoplasmic reticulum (ER). At pH 7 and 5, the peptide underwent a conformational change from random coil to alpha-helix upon addition of negatively charged phospholipids. Examination of the amide II band in the spectrum of the complex at pH 7 and pH 5 showed that in both cases, the N-H groups in the peptide backbone are largely protected from H/D exchange. Using polarized attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) measurements, the orientation of the alpha-helical portion of the peptide was found to be almost perpendicular with respect to the membrane plane at pH 7. However, at pH 5.0-5.4, the alpha-helix axis was preferentially oriented parallel to the membrane plane. The results suggest that at the neutral pH of the ER lumen, the peptide may insert into the membrane, while at the lower pH levels present in earlier endocytic compartments, the peptide would be less likely to traverse the bilayer. In summary, this putative signal peptide may not be able to cause a significant translocation of the A1 domain of Shiga toxin to the cytosol until it reaches the neutral pH of the ER compartment.

Amides↗

PubMed1989

Peptide signals for protein degradation within lysosomes.

In this article we summarize our findings concerning a pathway by which cytosolic proteins can be selectively taken up and degraded within lysosomes. Serum deprivation of cells in culture activates this pathway, and only proteins that contain peptide sequences related to KFERQ are degraded at an enhanced rate. Approximately 30% of intracellular proteins contain such peptide sequences, and we speculate about the physiological relevance of the selective degradation of these proteins in response to serum withdrawal. Several rat tissues also contain proteins with peptide sequences related to KFERQ, and the amount of these proteins is reduced in response to starvation. Finally, we present recent results suggesting that this selective uptake of cytosolic proteins by lysosomes is not through classical macroautophagy. Instead, the selective uptake appears to be similar to other protein sorting pathways such as protein translocation through the endoplasmic reticulum or protein import into mitochondria.

Amino Acid Sequence↗

PubMed2000

Cloning and sequencing of the gene encoding the candidate HtrA of Rickettsia typhi.

We screened a phage library of Rickettsia typhi with a polyclonal antiserum to clone genes which encode immunogenic proteins of R. typhi. Among several clones obtained, one clone codes for a 466-amino-acid protein similar to the heat-shock protein, HtrA. The deduced rickettsial HtrA contains a putative signal peptide sequence at the N-terminus, a serine protease-like domain, and two PDZ domains. The recombinant protein of rickettsial HtrA reacted with sera from patients with murine typhus and tsutsugamushi disease. We suggest that this gene and its recombinant protein would be valuable for the immunologic diagnosis of rickettsial diseases.

Amino Acid Sequence↗

PubMed1998

Characterization of the N-terminal targeting signal binding domain of the mitochondrial outer membrane receptor, Tom20.

hTom20 is an outer mitochondrial membrane receptor involved in protein translocation. The cytosolic domain (aa30-145) and selected truncated versions of this domain were overexpressed and purified to study the structure-function relationship of this protein. Our studies reveal that the secondary structure of the cytosolic domain is very resistant to unfolding by guanidine-HCl and urea and is stabilized mainly by hydrophobic interactions. However, the tertiary structure of the N-terminal targeting signal binding domain (aa30-90) is more flexible. The first 30 amino acids of the cytosolic domain (aa30-60) are involved in recognizing N-terminal targeting signals and in stabilizing the cytosolic domain on the lipid surface. Moreover, we show that specifically aa30-48 interact with the membrane surface; a construct containing aa48-145 will only bind to the membrane surface in the presence of an N-terminal targeting signal peptide. The C-terminal region of hTom20 (aa141-145) interacts with the N-terminal region of hTom20, helping to stabilize the proper conformation of the N-terminal targeting signal binding domain. Finally, hTom20 interacts with the N-terminal targeting signal of preornithine carbamyl transferase fused to dihydrofolate reductase very weakly (Kd = 8 microM), as would be expected if this interaction was the first in a series orchestrated by the import receptor complex to draw the targeted protein into the mitochondrion.

Circular Dichroism↗

PubMed1995

Conversion of a nonprocessed mitochondrial precursor protein into one that is processed by the mitochondrial processing peptidase.

Mitochondrial processing peptidase (MPP) cleaves the signal sequence from a variety of mitochondrial precursor proteins. A subset of mitochondrial proteins, including rhodanese and 3-oxoacyl-CoA thiolase, are imported into the matrix space, yet are not processed. Rhodanese signal peptide and translated protein were recognized by MPP, as both were inhibitors of processing. The signal peptide of precursor aldehyde dehydrogenase consists of a helix-linker-helix motif but when the RGP linker is removed, processing no longer occurs (Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D. G. (1993) J. Biol. Chem. 268, 19906-19914). Disruption of the helical signal sequence of rhodanese by the addition of the RGP linker did not allow cleavage to occur. However, addition of a putative cleavage site allowed the protein to be processed. The same cleavage site was added to 3-oxoacyl-CoA thiolase, but this protein was still not processed. Thiolase and linker-deleted aldehyde dehydrogenase signal peptides were poor inhibitors of MPP. It can be concluded that both a processing site and the structure surrounding this site are important for MPP recognition.

Acetyl-CoA C-Acyltransferase↗

PubMed2006

Protein targeting into the complex plastid of cryptophytes.

The cryptophyte Guillardia theta harbors a plastid surrounded by four membranes. This turns protein targeting of nucleus-encoded endosymbiont localized proteins into quite a challenge, as the respective precursors have to pass either all four membranes to reach the plastid stroma or only the outermost two membranes to enter the periplastidal compartment. Therefore two sets of nuclear-encoded proteins imported into the endosymbiont can be distinguished and their topogenic signals may serve as good indicators for studying protein targeting and subsequent transport across the outermost membranes of the cryptophyte plastid. We isolated genes encoding enzymes involved in two different biochemical pathways, both of which are predicted to be localized inside the periplastidal compartment, and compared their topogenic signals to those of precursor proteins for the plastid stroma, which are encoded on either the nucleus or the nucleomorph. By this and exemplary in vitro and in vivo analyses of the topogenic signal of one protein localized in the periplastidal compartment, we present new data implicating the mechanism of targeting and transport of proteins to and across the outermost plastid membranes. Furthermore, we demonstrate that one single, but conserved amino acid is the triggering key for the discrimination between nucleus-encoded plastid and periplastidal proteins.

Amino Acid Sequence↗

PubMed2006

Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display.

Even proteins that fold well in bacteria are frequently displayed poorly on filamentous phages. Low protein presentation on phage might be caused by premature cytoplasmic folding, leading to inefficient translocation into the periplasm. As translocation is an intermediate step in phage assembly, we tested the display levels of a range of proteins using different translocation pathways by employing different signal sequences. Directing proteins to the cotranslational signal recognition particle (SRP) translocation pathway resulted in much higher display levels than directing them to the conventional post-translational Sec translocation pathway. For example, the display levels of designed ankyrin-repeat proteins (DARPins) were improved up to 700-fold by simply exchanging Sec- for SRP-dependent signal sequences. In model experiments this exchange of signal sequences improved phage display from tenfold enrichment to >1,000-fold enrichment per phage display selection round. We named this method 'SRP phage display' and envision broad applicability, especially when displaying cDNA libraries or very stable and fast-folding proteins from libraries of alternative scaffolds.

Gene Expression Regulation, Bacterial↗

PubMed1995

The fates of proteins in cells.

Nascent polypeptide chains are in a dangerous situation as soon as they leave their place of birth, the channel of the large ribosomal subunit: more than 20 different pathways for the degradation of proteins exist in cells. Chaperones protect and guide the young protein molecules and support their correct foldings. Targeting signals direct the proteins to the organelles of their destination. The lysosome is the site of random degradation, while the proteasome is highly selective. Although these two organelles provide the most important pathways for the degradation of long- and short-lived proteins, other pathways with roles in deciding the fate of cellular proteins must also be considered.

Amino Acid Sequence↗

PubMed2006

Phage display of an intracellular carboxylesterase of Bacillus subtilis: comparison of Sec and Tat pathway export capabilities.

Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.

Adenosine Triphosphatases↗