scholarly journals A peptide corresponding to an export-defective mutant OmpA signal sequence with asparagine in the hydrophobic core is unable to insert into model membranes

1991 ◽  
Vol 266 (22) ◽  
pp. 14406-14412 ◽  
Author(s):  
D.W. Hoyt ◽  
L.M. Gierasch
Keyword(s):  
Signal Sequence ◽  
Model Membranes ◽  
Hydrophobic Core ◽  
Defective Mutant

scholarly journals Sec61p Contributes to Signal Sequence Orientation According to the Positive-Inside Rule

2004 ◽  
Vol 15 (3) ◽  
pp. 1470-1478 ◽  
Author(s):  
Veit Goder ◽  
Tina Junne ◽  
Martin Spiess
Keyword(s):  
Protein Targeting ◽  
Signal Sequence ◽  
Site Directed Mutagenesis ◽  
Diagnostic Model ◽  
Hydrophobic Core ◽  
Flanking Sequence ◽  
Charged Residues ◽  
Signal Anchor ◽  
A Charge

Protein targeting to the endoplasmic reticulum is mediated by signal or signal-anchor sequences. They also play an important role in protein topogenesis, because their orientation in the translocon determines whether their N- or C-terminal sequence is translocated. Signal orientation is primarily determined by charged residues flanking the hydrophobic core, whereby the more positive end is predominantly positioned to the cytoplasmic side of the membrane, a phenomenon known as the “positive-inside rule.” We tested the role of conserved charged residues of Sec61p, the major component of the translocon in Saccharomyces cerevisiae, in orienting signals according to their flanking charges by site-directed mutagenesis by using diagnostic model proteins. Mutation of R67, R74, or E382 in Sec61p reduced C-terminal translocation of a signal-anchor protein with a positive N-terminal flanking sequence and increased it for signal-anchor proteins with positive C-terminal sequences. These mutations produced a stronger effect on substrates with greater charge difference across the hydrophobic core of the signal. For some of the substrates, a charge mutation in Sec61p had a similar effect as one in the substrate polypeptides. Although these three residues do not account for the entire charge effect in signal orientation, the results show that Sec61p contributes to the positive-inside rule.

scholarly journals Glycine Residues in the Hydrophobic Core of the GspB Signal Sequence Route Export toward the Accessory Sec Pathway

2007 ◽  
Vol 189 (10) ◽  
pp. 3846-3854 ◽  
Author(s):  
Barbara A. Bensing ◽  
Ian R. Siboo ◽  
Paul M. Sullam
Keyword(s):  
Cell Surface ◽  
Signal Sequence ◽  
Human Platelets ◽  
Surface Glycoprotein ◽  
Simultaneous Increase ◽  
Hydrophobic Core ◽  
Cell Surface Glycoprotein ◽  
Sec Pathway ◽  
Amino Terminal ◽  
Sec System

ABSTRACT The Streptococcus gordonii cell surface glycoprotein GspB mediates high-affinity binding to distinct sialylated carbohydrate structures on human platelets and salivary proteins. GspB is glycosylated in the cytoplasm of S. gordonii and is then transported to the cell surface via a dedicated transport system that includes the accessory Sec components SecA2 and SecY2. The means by which the GspB preprotein is selectively recognized by the accessory Sec system have not been characterized fully. GspB has a 90-residue amino-terminal signal sequence that displays a traditional tripartite structure, with an atypically long amino-terminal (N) region followed by hydrophobic (H) and cleavage regions. In this report, we investigate the relative importance of the N and H regions of the GspB signal peptide for trafficking of the preprotein. The results show that the extended N region does not prevent export by the canonical Sec system. Instead, three glycine residues in the H region not only are necessary for export via the accessory Sec pathway but also interfere with export via the canonical Sec route. Replacement of the H-region glycine residues with helix-promoting residues led to a decrease in the efficiency of SecA2-dependent transport of the preprotein and a simultaneous increase in SecA2-independent translocation. Thus, the hydrophobic core of the GspB signal sequence is responsible primarily for routing towards the accessory Sec system.

scholarly journals Yeast carboxypeptidase Y can be translocated and glycosylated without its amino-terminal signal sequence.

1987 ◽  
Vol 104 (5) ◽  
pp. 1183-1191 ◽  
Author(s):  
E Blachly-Dyson ◽  
T H Stevens
Keyword(s):  
Secretory Pathway ◽  
Signal Sequence ◽  
Mutant Gene ◽  
Carboxypeptidase Y ◽  
Dependent Manner ◽  
Hydrophobic Core ◽  
Amino Terminal ◽  
Type Gene ◽  
Vacuolar Protein

We have constructed a series of mutations in the signal sequence of the yeast vacuolar protein carboxypeptidase Y (CPY), and have used pulse-chase radiolabeling and immunoprecipitation to examine the in vivo effects of these mutations on the entry of the mutant CPY proteins into the secretory pathway. We find that introduction of a negatively charged residue, aspartate, into the hydrophobic core of the signal sequence has no apparent effect on signal sequence function. In contrast, internal in-frame deletions within the signal sequence cause CPY to be synthesized as unglycosylated precursors. These are slowly and inefficiently converted to glycosylated precursors that are indistinguishable from the glycosylated forms produced from the wild-type gene. These precursors are converted to active CPY in a PEP4-dependent manner, indicating that they are correctly localized to the vacuole. Surprisingly, a deletion mutation that removes the entire CPY signal sequence has a similar effect: unglycosylated precursor accumulates in cells carrying this mutant gene, and greater than 10% of it is posttranslationally glycosylated. Thus, the amino-terminal signal sequence of CPY, while important for translocation efficiency, is not absolutely required for the translocation of this protein.

Discovery of functional motifs in h-regions of trypanosome signal sequences

2010 ◽  
Vol 426 (2) ◽  
pp. 135-145 ◽  
Author(s):  
Josh Duffy ◽  
Bhargavi Patham ◽  
Kojo Mensa-Wilmot
Keyword(s):  
Amino Acids ◽  
Signal Sequence ◽  
Bioinformatic Analysis ◽  
Surface Glycoprotein ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Hydrophobic Core ◽  
Signal Sequences ◽  
Peptide Motifs ◽  
Physiological Relevance

N-terminal signal peptides direct secretory proteins into the ER (endoplasmic reticulum) of eukaryotes or the periplasmic space of prokaryotes. A hydrophobic core (h-region) is important for signal sequence function; however, the mechanism of h-region action is not resolved. To gain new insight into signal sequences, bioinformatic analysis of h-regions from humans, Saccharomyces cerevisiae, Trypanosoma brucei and Escherichia coli was performed. Each species contains a unique set of peptide motifs (h-motifs) characterized by identity components (i.e. sequence of conserved amino acids) joined by spacers. Human h-motifs have four identity components, whereas those from the other species utilize three identity components. Example of h-motifs are human Hs3 {L-x(2)-[AGILPV]-L-x(0,2)-L}, S. cerevisiae Sc1 [L-x(0,2)-S-x(0,3)-A], T. brucei Tb2 {L-x(1,2)-L-[AILV]} and E. coli Ec1 [A-x(0,2)-L-x(0,3)-A]. The physiological relevance of h-motifs was tested with a T. brucei microsomal system for translocation of a VSG (variant surface glycoprotein)-117 signal peptide. Disruption of h-motifs by scrambling of sequences in h-regions produced defective signal peptides, although the hydrophobicity of the peptide was not altered. We conclude that: (i) h-regions harbour h-motifs, and are not random hydrophobic amino acids; (ii) h-regions from different species contain unique sets of h-motifs; and (iii) h-motifs contribute to the biological activity of ER signal peptides. h-Regions are ‘scaffolds’ in which functional h-motifs are embedded. A hypothetical model for h-motif interactions with a Sec61p protein translocon is presented.

scholarly journals P5A-ATPases control the ER translocation of Wnt for neuronal migration

2021 ◽  
Author(s):  
Tingting Li ◽  
Xiaoyan Yang ◽  
Zhigang Feng ◽  
Wang Nie ◽  
Yan Zou
Keyword(s):  
Neuronal Migration ◽  
Neuronal Development ◽  
Signal Sequence ◽  
Core Region ◽  
Hydrophobic Core ◽  
C Elegans ◽  
Wnt Proteins ◽  
Developmental Patterning ◽  
Er Targeting ◽  
Biochemical Analyses

Wnt family are conserved secreted proteins required for developmental patterning and tissue homeostasis. Research into the mechanisms that influence intracellular maturation and intercelluar signal transduction of Wnt proteins has proved fruitful. However, the knowledge of how Wnt enters into the endoplasmic reticulum (ER) for processing and secretion is still limited. Here we report that CATP-8/P5A-ATPase directs neuronal migration in C. elegans by controlling EGL-20/Wnt biogenesis. Our genetic and biochemical analyses demonstrate that CATP-8 control the ER targeting of EGL-20/Wnt through the hydrophobic core region in EGL-20 signal sequence. We further show that regulation of Wnt biogenesis by P5A-ATPase is conserved in human cells. These findings reveal physiological roles of P5A-ATPase in neuronal development and identify Wnt proteins as direct substrates of P5A-ATPase to be translocated into the ER.

scholarly journals Cloning and characterization of a cDNA for murine macrophage inflammatory protein (MIP), a novel monokine with inflammatory and chemokinetic properties.

1988 ◽  
Vol 167 (6) ◽  
pp. 1939-1944 ◽  
Author(s):  
G Davatelis ◽  
P Tekamp-Olson ◽  
S D Wolpe ◽  
K Hermsen ◽  
C Luedke ◽  
...  
Keyword(s):  
Sequence Data ◽  
Signal Sequence ◽  
Sequence Similarity ◽  
Alpha Helix ◽  
Phage Library ◽  
Hydrophobic Core ◽  
Inflammatory Protein ◽  
Characteristic Signal ◽  
Macrophage Inflammatory Protein

In the course of studies on cachectin/TNF being conducted in our laboratory, a novel macrophage product has been detected and characterized. Termed macrophage inflammatory protein or MIP, this protein appears to be an endogenous mediator of the inflammatory events induced by endotoxin. A cDNA cloned probe for this protein has been isolated from a lambda gt10 phage library prepared from poly(A)+ RNA obtained of endotoxin-induced RAW264.7 cells. The sequence codes for a 92 amino acid-long polypeptide, of which 69 amino acids correspond to the mature product. The sequence predicts a molecular weight of 7,889 and structural analysis of the protein indicates a characteristic signal sequence alpha-helix and a hydrophobic core. Sequence data also confirm no sequence similarity to any other protein listed in the Dayhoff data base.

scholarly journals Signal sequence-dependent function of the TRAM protein during early phases of protein transport across the endoplasmic reticulum membrane.

1996 ◽  
Vol 134 (1) ◽  
pp. 25-35 ◽  
Author(s):  
S Voigt ◽  
B Jungnickel ◽  
E Hartmann ◽  
T A Rapoport
Keyword(s):  
Protein Transport ◽  
Signal Sequence ◽  
Chain Complex ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Hydrophobic Core ◽  
Sequence Dependent ◽  
Nascent Chain ◽  
Er Membrane

Cotranslational translocation of proteins across the mammalian ER membrane involves, in addition to the signal recognition particle receptor and the Sec61p complex, the translocating chain-associating membrane (TRAM) protein, the function of which is still poorly understood. Using reconstituted proteoliposomes, we show here that the translocation of most, but not all, secretory proteins requires the function of TRAM. Experiments with hybrid proteins demonstrate that the structure of the signal sequence determines whether or not TRAM is needed. Features that distinguish TRAM-dependent and -independent signal sequences include the length of their charged, NH2-terminal region and the structure of their hydrophobic core. In cases where TRAM is required for translocation, it is not needed for the initial interaction of the ribosome/nascent chain complex with the ER membrane but for a subsequent step inside the membrane in which the nascent chain is inserted into the translocation site in a protease-resistant manner. Thus, TRAM functions in a signal sequence-dependent manner at a critical, early phase of the translocation process.

scholarly journals Molecular components of the signal sequence that function in the initiation of protein export.

1982 ◽  
Vol 95 (3) ◽  
pp. 689-696 ◽  
Author(s):  
S D Emr ◽  
T J Silhavy
Keyword(s):  
Amino Acids ◽  
Signal Sequence ◽  
Protein Localization ◽  
Helical Conformation ◽  
Hydrophobic Core ◽  
Selection Procedures ◽  
Sequence Characterization ◽  
Mutant Strains ◽  
Molecular Components

We are studying the mechanism by which the LamB protein is exported to the outer membrane of Escherichia coli. Using two selection procedures based on gene fusions, we have identified a number of mutations that cause alterations in the LamB signal sequence. Characterization of the mutant strains revealed that although many such mutations block LamB export to greater than 95%, others have essentially no effect. These results allow an analysis of the functions performed by the various molecular components of the signal sequence. Our results suggest that a critical subset of four amino acids is contained within the central hydrophobic core of the LamB signal sequence. If this core can assume an alpha-helical conformation, these four amino acids comprise a recognition site that interacts with a component of the cellular export machinery. Since mechanisms of protein localization appear to have been conserved during evolution, the principles established by these results should be applicable to similar studies in eukaryotic cells.

scholarly journals Regulation of the Ribosome–Membrane Junction at Early Stages of Presecretory Protein Translocation in the Mammalian Endoplasmic Reticulum

1997 ◽  
Vol 139 (7) ◽  
pp. 1697-1708 ◽  
Author(s):  
Christopher V. Nicchitta ◽  
Tianli Zheng
Keyword(s):  
Endoplasmic Reticulum ◽  
Fusion Protein ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Secretory Protein ◽  
Membrane Topology ◽  
Hydrophobic Core ◽  
Conducting Channel ◽  
Nascent Chain ◽  
Potential Mode

A series of fusion protein constructs were designed to investigate the contribution of secretory nascent chains to regulation of the ribosome–membrane junction in the mammalian endoplasmic reticulum. As a component of these studies, the membrane topology of the signal sequence was determined at stages of protein translocation immediately after targeting and before signal sequence cleavage. Truncated translation products were used to delimit the analysis to defined stages of translocation. In a study of secretory protein precursors, formation of a protease-resistant ribosome–membrane junction, currently thought to define the pathway of the translocating nascent chain, was observed to be precursor- and stage-dependent. Analysis of the binding of early intermediates indicated that the nascent chain was bound to the membrane independent of the ribosome, and that the binding was predominately electrostatic. The membrane topology of the signal sequence was determined as a function of the stage of translocation, and was found to be identical for all assayed intermediates. Unexpectedly, the hydrophobic core of the signal sequence was observed to be accessible to the cytosolic face of the membrane at stages of translocation immediately after targeting as well as stages before signal sequence cleavage. Removal of the ribosome from bound intermediates did not disrupt subsequent translocation, suggesting that the active state of the protein-conducting channel is maintained in the absence of the bound ribosome. A model describing a potential mode of regulation of the ribosome–membrane junction by the nascent chain is presented.

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