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Results for “Protein Sorting Signals”

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At least 343 records · Page 19Linked to original sources

PubMed2005

Use of thioredoxin as a reporter to identify a subset of Escherichia coli signal sequences that promote signal recognition particle-dependent translocation.

We have previously reported that the DsbA signal sequence promotes efficient, cotranslational translocation of the cytoplasmic protein thioredoxin-1 via the bacterial signal recognition particle (SRP) pathway. However, two commonly used signal sequences, those of PhoA and MalE, which promote export by a posttranslational mechanism, do not export thioredoxin. We proposed that this difference in efficiency of export was due to the rapid folding of thioredoxin in the cytoplasm; cotranslational export by the DsbA signal sequence avoids the problem of cytoplasmic folding (C. F. Schierle, M. Berkmen, D. Huber, C. Kumamoto, D. Boyd, and J. Beckwith, J. Bacteriol. 185:5706-5713, 2003). Here, we use thioredoxin as a reporter to distinguish SRP-dependent from non-SRP-dependent cleavable signal sequences. We screened signal sequences exhibiting a range of hydrophobicity values based on a method that estimates hydrophobicity. Successive iterations of screening and refining the method defined a threshold hydrophobicity required for SRP recognition. While all of the SRP-dependent signal sequences identified were above this threshold, there were also a few signal sequences above the threshold that did not utilize the SRP pathway. These results suggest that a simple measure of the hydrophobicity of a signal sequence is an important but not a sufficient indicator for SRP recognition. In addition, by fusing a number of both classes of signal sequences to DsbA, we found that DsbA utilizes an SRP-dependent signal sequence to achieve efficient export to the periplasm. Our results suggest that those proteins found to be exported by SRP-dependent signal sequences may require this mode of export because of their tendency to fold rapidly in the cytoplasm.

Blotting, Western↗

PubMed1996

Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins.

Extracellular growth and differentiation factors induce changes in gene expression in the nucleus by initiating a series of protein associations that alter the subcellular localization of intracellular signaling proteins. Initial events involve receptor homo- or heterodimerization and subsequent recruitment of cytosolic signaling proteins to the inner leaflet of the plasma membrane. Intermediate events involve the translocation of proteins into the nucleus. Late events involve the recruitment of transcriptional activators to the vicinity of specific genes in the nucleus, resulting in increased gene transcription. The ability to induce signals at each of these three phases of signaling pathways is illustrated by the use of a heterodimeric chemical inducer of dimerization that causes a proximal relationship between two different target proteins.

Animals↗

PubMed2003

Signal sequences initiate the pathway of maturation in the endoplasmic reticulum lumen.

An interaction between an N-terminal signal sequence and the translocon leads to the initiation of protein translocation into the endoplasmic reticulum lumen. Subsequently, folding and modification of the substrate rapidly ensue. The close temporal coordination of these processes suggests that they may be structurally and functionally coordinated as well. Here we show that information encoded in the hydrophobic domain of a signal sequence influences the timing and efficiency of at least two steps in maturation, namely N-linked glycosylation and signal sequence cleavage. We demonstrate that these consequences correlate with and likely stem from the nature of the initial association made between the signal sequence and the translocon during the initiation of translocation. We propose a model by which these maturational events are controlled by the signal sequence-translocon interaction. Our work demonstrates that the pathway taken by a nascent chain through post-translational maturation depends on information encoded in its signal sequence.

Amino Acid Sequence↗

PubMed1990

A novel pathway for secretory proteins?

In eukaryotes, most proteins which are transported to the extracellular space, into mitochondria or into chloroplasts are synthesized as precursor polypeptides containing cleavable N-terminal signal or targeting sequences. We have searched the literature for proteins that are exported from the cytosol without being proteolytically processed. Some of these proteins contain uncleaved signal or targeting sequences. However, among secretory proteins there is a class that does not possess hydrophobic signal sequences and appears to leave the cell by a secretory pathway clearly distinct from the classical route through the endoplasmic reticulum and Golgi apparatus.

Acylation↗

PubMed2001

Quality control in the endoplasmic reticulum: PDI mediates the ER retention of unassembled procollagen C-propeptides.

Quality control within the endoplasmic reticulum (ER) is thought to be mediated by the interaction of a folding protein with one or several resident ER proteins [1]. Protein disulphide isomerase (PDI) is one such ER resident protein that has been previously shown to interact with proteins during their folding and assembly pathways [2, 3]. It has been assumed that, as a consequence of this interaction, unassembled proteins are retained within the ER. Here, we experimentally show that this is indeed the case. We have taken advantage of our previous finding that PDI interacts with procollagen chains early on in their assembly pathway [2] to address the role of this protein in directly retaining unassembled chains within the ER. Our experimental approach involved expressing individual C-propeptide domains from different procollagen chains in mammalian cells and determining the ability of these domains to interact with PDI and to be secreted. The C-propeptide from the proalpha2(I) chain was retained within the cell, where it formed a complex with PDI. Conversely, the C-propeptide from the proalpha1(III) chain did not form a complex with PDI and was secreted. Both domains were secreted, however, from a stable cell line expressing a secreted form of PDI lacking its ER retrieval signal. Hence, we have demonstrated directly that the intracellular retention of one substrate for ER quality control is due to an interaction with PDI.

Cell Line↗

PubMed2003

A profile hidden Markov model for signal peptides generated by HMMER.

SUMMARY: Although the HMMER package is widely used to produce profile hidden Markov models (profile HMMs) for protein domains, it has been difficult to create a profile HMM for signal peptides. Here we describe an approach for building a complex model of eukaryotic signal peptides by the standard HMMER package. Signal peptide prediction with this model gives a 95.6% sensitivity and 95.7% specificity. AVAILABILITY: The profile HMM for signal peptides, data sets, and the scripts for analyzing data are available for non-commercial use at http://share.gene.com/.

Amino Acid Sequence↗

PubMed1991

[Evolution of regulatory proteins].

A concept of the evolution of signal molecules and their receptors is proposed. The formation of novel regulatory proteins by way of gene fusion and gene shuffling and the role of these processes in metabolic integration are considered. The different receptor variants may be due to alternative splicing which is regulated in a tissue-specific manner.

Biological Evolution↗

PubMed2004

Demonstration of a specific Escherichia coli SecY-signal peptide interaction.

Protein translocation in Escherichia coli is initiated by the interaction of a preprotein with the membrane translocase composed of a motor protein, SecA ATPase, and a membrane-embedded channel, the SecYEG complex. The extent to which the signal peptide region of the preprotein plays a role in SecYEG interactions is unclear, in part because studies in this area typically employ the entire preprotein. Using a synthetic signal peptide harboring a photoaffinity label in its hydrophobic core, we examined this interaction with SecYEG in a detergent micellar environment. The signal peptide was found to specifically bind SecY in a saturable manner and at levels comparable to those that stimulate SecA ATPase activity. Chemical and proteolytic cleavage of cross-linked SecY and analysis of the signal peptide adducts indicate that the binding was primarily to regions of the protein containing transmembrane domains seven and two. The signal peptide-SecY interaction was affected by the presence of SecA and nucleotides in a manner consistent with the transfer of signal peptide to SecY upon nucleotide hydrolysis at SecA.

Adenosine Triphosphatases↗

PubMed2000

Assembly, target-signaling and intracellular transport of tyrosinase gene family proteins in the initial stage of melanosome biogenesis.

Assembly, target-signaling and transport of tyrosinase gene family proteins at the initial stage of melanosome biogenesis are reviewed based on our own discoveries. Melanosome biogenesis involves four stages of maturation with distinct morphological and biochemical characteristics that reflect distinct processes of the biosynthesis of structural and enzymatic proteins, subsequent structural organization and melanin deposition occurring in these particular cellular compartments. The melanosomes share many common biological properties with the lysosomes. The stage I melanosomes appear to be linked to the late endosomes. Most of melanosomal proteins are glycoproteins that should be folded or assembled correctly in the ER through interaction with calnexin, a chaperone associated with melanogenesis. These melanosomal glycoproteins are then accumulated in the trans Golgi network (TGN) and transported to the melanosomal compartment. During the formation of transport vesicles, coat proteins assemble on the cytoplasmic face of TGN to select their cargos by interacting directly or indirectly with melanosomal glycoproteins to be transported. Adapter protein-3 (AP-3) is important for intracellular transport of tyrosinase gene family proteins from TGN to melanosomes. Tyrosinase gene family proteins possess a di-leucine motif in their cytoplasmic tail, to which AP-3 appears to bind. Thus, the initial cascade of melanosome biogenesis is regulated by several factors including: 1) glycosylation of tyrosinase gene family proteins and their correct folding and assembly within ER and Golgi, and 2) supply of specific signals necessary for intracellular transport of these glycoproteins by vesicles from Golgi to melanosomes.

Amino Acid Sequence↗

PubMed2003

The M-PMV cytoplasmic targeting-retention signal directs nascent Gag polypeptides to a pericentriolar region of the cell.

Intracytoplasmic protein targeting in mammalian cells is critical for organelle function as well as virus assembly, but the signals that mediate it are poorly defined. We show here that Mason-Pfizer monkey virus specifically targets Gag precursor proteins to the pericentriolar region of the cytoplasm in a microtubule dependent process through interactions between a short peptide signal, known as the cytoplasmic targeting-retention signal, and the dynein/dynactin motor complex. The Gag molecules are concentrated in pericentriolar microdomains, where they assemble to form immature capsids. Depletion of Gag from this region by cycloheximide treatment, coupled with the presence of ribosomal clusters that are in close vicinity to the assembling capsids, suggests that the dominant N-terminal cytoplasmic targeting-retention signal functions in a cotranslational manner. Transport of the capsids out of the pericentriolar assembly site requires the env-gene product, and a functional vesicular transport system. A single point mutation that renders the cytoplasmic targeting-retention signal defective abrogates pericentriolar targeting of Gag molecules. Thus the previously defined cytoplasmic targeting-retention signal appears to act as a cotranslational intracellular targeting signal that concentrates Gag proteins at the centriole for assembly of capsids.

Animals↗

PubMed1985

A former amino terminal signal sequence engineered to an internal location directs translocation of both flanking protein domains.

To determine whether a functional amino terminal signal sequence can be active at an internal position, a hybrid gene was constructed in which the entire coding region of bovine preprolactin cDNA was inserted into chimpanzee alpha-globin cDNA 109 codons downstream from the initiation codon of globin. When RNA synthesized in vitro from this plasmid (pSPGP1) was translated in the rabbit reticulocyte cell-free system, a 32-kD protein was produced that was both prolactin and globin immunoreactive. When microsomal membranes were present during translation (but not when added posttranslationally), a 26-kD and a 14-kD product were also observed. By immunoreactivity and electrophoretic mobility, the 26-kD protein was identical to mature prolactin, and the 14-kD protein appeared to be the globin domain with the prolactin signal sequence attached at its carboxy terminus. From (a) posttranslational proteolysis in the presence and absence of detergent, (b) sedimentation of vesicles in the presence and absence of sodium carbonate pH 11.5, and (c) N-linked glycosylation of the globin-immunoreactive fragment after insertion of an Asn-X-Ser N-linked glycosylation site into the globin coding region of pSPGP1, it appears that all of the 26-kD and some of the 14-kD products, but none of the 32-kD precursor, have been translocated to the lumen of the membrane vesicles. Thus, when engineered to an internal position, the prolactin signal sequence is able to translocate both flanking protein domains. These data have implications for the understanding of translocation of proteins across the membrane of the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed1993

Switch in antiviral specificity of a GTPase upon translocation from the cytoplasm to the nucleus.

The Mx2 protein of rats is a cytoplasmic GTPase that protects cells against vesicular stomatitis virus but not against influenza virus. Since vesicular stomatitis virus replicates in the cytoplasm and influenza virus replicates in the nucleus, it was possible that the antiviral specificity of rat Mx2 protein was determined solely by the protein's subcellular localization. Here, we found that, indeed, rat Mx2 protein lost its anti-vesicular stomatitis virus activity and gained anti-influenza virus activity when it was directed to the nucleus by way of a foreign nuclear-transport signal appended to its amino terminus. These data show that rat Mx2 protein possesses an antiviral activity that is revealed only when the protein is shuttled to the nucleus.

3T3 Cells↗

PubMed2001

In situ hybridization screen in zebrafish for the selection of genes encoding secreted proteins.

An in situ hybridization expression screen using a signal sequence trap system has been conducted in zebrafish to isolate cDNAs that encode secreted proteins. Random clones (secreted expressed sequence tags; sESTs) were sequenced from zebrafish embryonic (18-24 hr postfertilization) and adult kidney libraries. From the two RNA sources, 627 random sEST cDNAs were identified as being homologous or identical to known genes and 166 clones encode currently unidentified genes. The sESTs represent a broad range of enzymes and other regulatory molecules. Whole-mount in situ hybridization analysis was carried out by using antisense probes generated from 244 selected sESTs, and a range of expression patterns was obtained. Genetic mapping undertaken with sEST sequences demonstrated that assignment of map position was attainable by using 5' primers. The signal sequence trap system used in this work has yielded a range of cDNAs that encode secreted proteins and, together with analysis of patterns of expression and genetic mapping, has the potential to facilitate analysis of signaling pathways central to development and physiology.

Animals↗

PubMed2001

An ER retention signal explains differences in surface expression of NMDA and AMPA receptor subunits.

The molecular mechanisms that control the surface expression of NMDA receptors (NMDARs) and AMPA receptors (AMPARs) are unknown. To determine the role of the intracellular C-terminal tails of glutamate receptor subunits in the synaptic targeting of AMPARs and NMDARs, we fused the tails of the AMPAR subunits, GluR1 and GluR2, and the NMDAR subunit, NR1, to the human T lymphocyte membrane protein CD8 and expressed these constructs in HEK293 cells and cultured hippocampal neurons. The GluR1 and GluR2 fusion proteins exhibited robust surface expression in the plasma membrane of neurons at synapses as did CD8 alone. In contrast, the NR1 fusion protein was retained intracellularly in both HEK293 cells and neurons because of the presence of an ER retention signal in the C1 cassette. This ER retention signal was overridden either by the addition of a PDZ domain-binding motif or by mimicking phosphorylation at a site adjacent to the retention signal. These results provide further evidence that the intracellular trafficking of AMPAR and NMDAR subunits are regulated independently at least in part because of differences in the protein-protein interactions of their intracellular C-terminal tails.

Animals↗

PubMed2001

Molecular cloning of a novel ubiquitin-like protein, UBIN, that binds to ER targeting signal sequences.

To identify proteins that interact with HSP47, an endoplasmic reticulum (ER)-resident molecular chaperone, a yeast two-hybrid screening was performed using mouse full-length HSP47 including an N-terminal signal sequence as a bait. Analysis of several positive clones led to the identification and cloning of a novel gene, ubin, encoding a ubiquitin-like protein. Unlike other ubiquitin-like proteins, UBIN was shown to interact with signal sequences of various secretory and ER-luminal proteins, including HSP47, but not interact with signal sequences of mitochondrial targeting in two-hybrid system. The possible function of UBIN will be discussed with regards to novel characteristics of binding to signal sequences for ER targeting.

3T3 Cells↗

PubMed2005

The conformation of a signal peptide bound by Escherichia coli preprotein translocase SecA.

To understand the structural nature of signal sequence recognition by the preprotein translocase SecA, we have characterized the interactions of a signal peptide corresponding to a LamB signal sequence (modified to enhance aqueous solubility) with SecA by NMR methods. One-dimensional NMR studies showed that the signal peptide binds SecA with a moderately fast exchange rate (Kd approximately 10(-5) m). The line-broadening effects observed from one-dimensional and two-dimensional NMR spectra indicated that the binding mode does not equally immobilize all segments of this peptide. The positively charged arginine residues of the n-region and the hydrophobic residues of the h-region had less mobility than the polar residues of the c-region in the SecA-bound state, suggesting that this peptide has both electrostatic and hydrophobic interactions with the binding pocket of SecA. Transferred nuclear Overhauser experiments revealed that the h-region and part of the c-region of the signal peptide form an alpha-helical conformation upon binding to SecA. One side of the hydrophobic core of the helical h-region appeared to be more strongly bound in the binding pocket, whereas the extreme C terminus of the peptide was not intimately involved. These results argue that the positive charges at the n-region and the hydrophobic helical h-region are the selective features for recognition of signal sequences by SecA and that the signal peptide-binding site on SecA is not fully buried within its structure.

Adenosine Triphosphatases↗

PubMed2000

A Drosophila IkappaB kinase complex required for Relish cleavage and antibacterial immunity.

Here we report the identification of a Drosophila IkappaB kinase complex containing DmIKKbeta and DmIKKgamma, homologs of the human IKKbeta and IKKgamma proteins. We show that this complex is required for the signal-dependent cleavage of Relish, a member of the Rel family of transcriptional activator proteins, and for the activation of antibacterial immune response genes. In addition, we find that the activated DmIKK complex, as well as recombinant DmIKKbeta, can phosphorylate Relish in vitro. Thus, we propose that the Drosophila IkappaB kinase complex functions, at least in part, by inducing the proteolytic cleavage of Relish. The N terminus of Relish then translocates to the nucleus and activates the transcription of antibacterial immune response genes. Remarkably, this Drosophila IkappaB kinase complex is not required for the activation of the Rel proteins Dif and Dorsal through the Toll signaling pathway, which is essential for antifungal immunity and dorsoventral patterning during early development. Thus, a yet to be identified IkappaB kinase complex must be required for Rel protein activation via the Toll signaling pathway.

Animals↗

PubMed2004

Many fingers on the mitotic trigger: post-translational regulation of the Cdc25C phosphatase.

The mitotic inducer Cdc25 phosphatase controls the activation of Cdc2/cyclin B protein kinase and entry into mitosis in eukaryotic cells. Cdc25C is highly regulated by multiple post-translational modifications within its N-terminal regulatory domain and site-specific protein interactions. Phosphorylation of one inhibitory site targeted by multiple kinases determines the timing of Cdc25C activation and arrests cells in G2 in response to checkpoint, stress, developmental and extracellular signals. In mitosis, phosphorylation of several Ser/Thr residues and Pin1-catalysed peptidyl-proline isomerisation produces activation. Phosphorylation of one activating site is antagonistic to the proximal inhibitory site and maintains Cdc25C activity during mitosis. Phosphorylation and interacting proteins also modulate the nuclear import and export signals on Cdc25C, inducing dramatic changes in its localisation within the cell. Thus, the regulation of Cdc25C activity and localization integrates multiple signals that govern the decision to enter mitosis.

Active Transport, Cell Nucleus↗