Conformations of wild-type and mutant OmpA signal sequences in membrane mimetic environments

Peptides ◽  
1992 ◽  
pp. 265-267
Author(s):  
Josep Rizo ◽  
Francisco Blanco ◽  
Bostjan Kobe ◽  
Martha D. Bruch ◽  
David W. Hoyt ◽  
...  
Keyword(s):  
Wild Type ◽  
Signal Sequences ◽  
Membrane Mimetic

scholarly journals Interaction of wild-type signal sequences and their charged variants with model and natural membranes

1993 ◽  
Vol 293 (1) ◽  
pp. 43-49 ◽  
Author(s):  
N M Rao ◽  
R Nagaraj
Keyword(s):  
Amino Acids ◽  
Synthetic Peptides ◽  
Model Membranes ◽  
Signal Peptides ◽  
Wild Type ◽  
Signal Sequences ◽  
Hydrophobic Region ◽  
Phospholipases A2 ◽  
Charged Amino Acids

The interaction of synthetic peptides corresponding to wild-type signal sequences, and their mutants having charged amino acids in the hydrophobic region, with model and natural membranes has been studied. At high peptide concentrations, i.e. low lipid/peptide ratios, the signal peptides cause release of carboxyfluorescein (CF) from model membranes with lipid compositions corresponding to those of translocation-competent as well as translocation-incompetent membranes. Interestingly, mutant sequences, which were non-functional in vivo, caused considerable release of CF compared with the wild-type sequences. Both wild-type and mutant signal sequences perturb model membranes even at lipid/peptide ratios of 1000:1, as indicated by the activities of phospholipases A2, C and D. These studies indicate that such mutant signals are non-functional not because of their inability to interact with membranes, but due to defective targeting to the membrane. The signal peptides inhibit phospholipase C activity in microsomes, uncouple oxidative phosphorylation in mitochondria and increase K+ efflux from erythrocytes, and one of the mutant sequences is a potent degranulator of the mast cells. Both wild-type and mutant signal sequences have the ability to perturb vesicles of various lipid compositions. With respect to natural membranes, the peptides do not show any bias towards translocation-competent membranes.

scholarly journals Dependency of Coxiella burnetii Type 4B Secretion on the Chaperone IcmS

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Charles L. Larson ◽  
Paul A. Beare ◽  
Robert A. Heinzen
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Legionella Pneumophila ◽  
Q Fever ◽  
Secretion System ◽  
Host Cells ◽  
Growth Defect ◽  
Wild Type ◽  
Signal Sequences ◽  
Content Type

ABSTRACT Macrophage parasitism by Coxiella burnetii, the cause of human Q fever, requires the translocation of proteins with effector functions directly into the host cell cytosol via a Dot/Icm type 4B secretion system (T4BSS). Secretion by the analogous Legionella pneumophila T4BSS involves signal sequences within the C-terminal and internal domains of effector proteins. The cytoplasmic chaperone pair IcmSW promotes secretion and binds internal sites distinct from signal sequences. In the present study, we investigated requirements of C. burnetii IcmS for host cell parasitism and effector translocation. A C. burnetii icmS deletion mutant (ΔicmS) exhibited impaired replication in Vero epithelial cells, deficient formation of the Coxiella-containing vacuole, and aberrant T4BSS secretion. Three secretion phenotypes were identified from a screen of 50 Dot/Icm substrates: IcmS dependent (secreted by only wild-type bacteria), IcmS independent (secreted by both wild-type and ΔicmS bacteria), or IcmS inhibited (secreted by only ΔicmS bacteria). Secretion was assessed for N-terminal or C-terminal truncated forms of CBU0794 and CBU1525. IcmS-inhibited secretion of CBU1525 required a C-terminal secretion signal whereas IcmS-dependent secretion of CBU0794 was directed by C-terminal and internal signals. Interchange of the C-terminal 50 amino acids of CBU0794 and CBU1525 revealed that sites within the C terminus regulate IcmS dependency. Glutathione S-transferase-tagged IcmSW bound internal sequences of IcmS-dependent and -inhibited substrates. Thus, the growth defect of the C. burnetii ΔicmS strain is associated with a loss of T4BSS chaperone activity that both positively and negatively regulates effector translocation. IMPORTANCE The intracellular pathogen Coxiella burnetii employs a type 4B secretion system (T4BSS) that promotes growth by translocating effectors of eukaryotic pathways into host cells. T4BSS regulation modeled in Legionella pneumophila indicates IcmS facilitates effector translocation. Here, we characterized type 4B secretion by a Coxiella ΔicmS mutant that exhibits intracellular growth defects. T4BSS substrates demonstrated increased, equivalent, or decreased secretion by the ΔicmS mutant relative to wild-type Coxiella. Similar to the Legionella T4BSS, IcmS dependency in Coxiella was determined by C-terminal and/or internal secretion signals. However, IcmS inhibited secretion of some effectors by Coxiella that were previously shown to be translocated by Legionella. Thus, Coxiella has a unique IcmS regulatory mechanism that both positively and negatively regulates T4BSS export.

scholarly journals Comparison of helix stability in wild-type and mutant LamB signal sequences.

1990 ◽  
Vol 265 (7) ◽  
pp. 3851-3858
Author(s):  
M D Bruch ◽  
L M Gierasch
Keyword(s):  
Wild Type ◽  
Signal Sequences ◽  
Helix Stability

Strand breaks without DNA rearrangement in V (D)J recombination

1991 ◽  
Vol 11 (6) ◽  
pp. 3155-3162
Author(s):  
E A Hendrickson ◽  
V F Liu ◽  
D T Weaver
Keyword(s):  
B Cells ◽  
Signal Sequence ◽  
Cell Receptor ◽  
Retroviral Vectors ◽  
Sequence Motifs ◽  
Wild Type ◽  
Severe Combined Immune Deficiency ◽  
Signal Sequences ◽  
Strand Breaks ◽  
Recombination Pathway

Somatic gene rearrangement of immunoglobulin and T-cell receptor genes [V(D)J recombination] is mediated by pairs of specific DNA sequence motifs termed signal sequences. In experiments described here, retroviral vectors containing V(D)J rearrangement cassettes in which the signal sequences had been altered were introduced into wild-type and scid (severe combined immune deficiency) pre-B cells and used to define intermediates in the V(D)J recombination pathway. The scid mutation has previously been shown to deleteriously affect the V(D)J recombination process. Cassettes containing a point mutation in one of the two signal sequences inhibited rearrangement in wild-type cells. In contrast, scid cells continued to rearrange these cassettes with the characteristic scid deletional phenotype. Using these mutated templates, we identified junctional modifications at the wild-type signal sequences that had arisen from strand breaks which were not associated with overall V(D)J rearrangements. Neither cell type was able to rearrange constructs which contained only a single, nonmutated, signal sequence. In addition, scid and wild-type cell lines harboring cassettes with mutations in both signal sequences did not undergo rearrangement, suggesting that at least one functional signal sequence was required for all types of V(D)J recombination events. Analysis of these signal sequence mutations has provided insights into intermediates in the V(D)J rearrangement pathway in wild-type and scid pre-B cells.

scholarly journals Strand breaks without DNA rearrangement in V (D)J recombination.

1991 ◽  
Vol 11 (6) ◽  
pp. 3155-3162 ◽  
Author(s):  
E A Hendrickson ◽  
V F Liu ◽  
D T Weaver
Keyword(s):  
B Cells ◽  
Signal Sequence ◽  
Cell Receptor ◽  
Retroviral Vectors ◽  
Sequence Motifs ◽  
Wild Type ◽  
Severe Combined Immune Deficiency ◽  
Signal Sequences ◽  
Strand Breaks ◽  
Recombination Pathway

Somatic gene rearrangement of immunoglobulin and T-cell receptor genes [V(D)J recombination] is mediated by pairs of specific DNA sequence motifs termed signal sequences. In experiments described here, retroviral vectors containing V(D)J rearrangement cassettes in which the signal sequences had been altered were introduced into wild-type and scid (severe combined immune deficiency) pre-B cells and used to define intermediates in the V(D)J recombination pathway. The scid mutation has previously been shown to deleteriously affect the V(D)J recombination process. Cassettes containing a point mutation in one of the two signal sequences inhibited rearrangement in wild-type cells. In contrast, scid cells continued to rearrange these cassettes with the characteristic scid deletional phenotype. Using these mutated templates, we identified junctional modifications at the wild-type signal sequences that had arisen from strand breaks which were not associated with overall V(D)J rearrangements. Neither cell type was able to rearrange constructs which contained only a single, nonmutated, signal sequence. In addition, scid and wild-type cell lines harboring cassettes with mutations in both signal sequences did not undergo rearrangement, suggesting that at least one functional signal sequence was required for all types of V(D)J recombination events. Analysis of these signal sequence mutations has provided insights into intermediates in the V(D)J rearrangement pathway in wild-type and scid pre-B cells.

scholarly journals A gating motif in the translocation channel sets the hydrophobicity threshold for signal sequence function

2012 ◽  
Vol 199 (6) ◽  
pp. 907-918 ◽  
Author(s):  
Steven F. Trueman ◽  
Elisabet C. Mandon ◽  
Reid Gilmore
Keyword(s):  
Amino Acid ◽  
Structural Transition ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Bioinformatic Analysis ◽  
Critical Event ◽  
Wild Type ◽  
Polar Amino Acid ◽  
Signal Sequences ◽  
Channel Opening

A critical event in protein translocation across the endoplasmic reticulum is the structural transition between the closed and open conformations of Sec61, the eukaryotic translocation channel. Channel opening allows signal sequence insertion into a gap between the N- and C-terminal halves of Sec61. We have identified a gating motif that regulates the transition between the closed and open channel conformations. Polar amino acid substitutions in the gating motif cause a gain-of-function phenotype that permits translocation of precursors with marginally hydrophobic signal sequences. In contrast, hydrophobic substitutions at certain residues in the gating motif cause a protein translocation defect. We conclude that the gating motif establishes the hydrophobicity threshold for functional insertion of a signal sequence into the Sec61 complex, thereby allowing the wild-type translocation channel to discriminate between authentic signal sequences and the less hydrophobic amino acid segments in cytosolic proteins. Bioinformatic analysis indicates that the gating motif is conserved between eubacterial and archaebacterial SecY and eukaryotic Sec61.

scholarly journals Identification and Characterization of a Gain-of-Function RAG-1 Mutant

2006 ◽  
Vol 26 (12) ◽  
pp. 4712-4728 ◽  
Author(s):  
Aleksei N. Kriatchko ◽  
Dirk K. Anderson ◽  
Patrick C. Swanson
Keyword(s):  
Binding Activity ◽  
Strand Transfer ◽  
Wild Type ◽  
Gain Of Function ◽  
Signal Sequences ◽  
Cleavage Activity ◽  
Dna Binding Activity ◽  
Rag Proteins ◽  
Rag 1

ABSTRACT RAG-1 and RAG-2 initiate V(D)J recombination by cleaving DNA at recombination signal sequences through sequential nicking and transesterification reactions to yield blunt signal ends and coding ends terminating in a DNA hairpin structure. Ubiquitous DNA repair factors then mediate the rejoining of broken DNA. V(D)J recombination adheres to the 12/23 rule, which limits rearrangement to signal sequences bearing different lengths of DNA (12 or 23 base pairs) between the conserved heptamer and nonamer sequences to which the RAG proteins bind. Both RAG proteins have been subjected to extensive mutagenesis, revealing residues required for one or both cleavage steps or involved in the DNA end-joining process. Gain-of-function RAG mutants remain unidentified. Here, we report a novel RAG-1 mutation, E649A, that supports elevated cleavage activity in vitro by preferentially enhancing hairpin formation. DNA binding activity and the catalysis of other DNA strand transfer reactions, such as transposition, are not substantially affected by the RAG-1 mutation. However, 12/23-regulated synapsis does not strongly stimulate the cleavage activity of a RAG complex containing E649A RAG-1, unlike its wild-type counterpart. Interestingly, wild-type and E649A RAG-1 support similar levels of cleavage and recombination of plasmid substrates containing a 12/23 pair of signal sequences in cell culture; however, E649A RAG-1 supports about threefold more cleavage and recombination than wild-type RAG-1 on 12/12 plasmid substrates. These data suggest that the E649A RAG-1 mutation may interfere with the RAG proteins' ability to sense 12/23-regulated synapsis.

scholarly journals Escherichia coli SecG Is Required for Residual Export Mediated by Mutant Signal Sequences and for SecY-SecE Complex Stability

2014 ◽  
Vol 197 (3) ◽  
pp. 542-552 ◽  
Author(s):  
Dominique Belin ◽  
Giuseppe Plaia ◽  
Yasmine Boulfekhar ◽  
Filo Silva
Keyword(s):  
Critical Role ◽  
Standard Laboratory ◽  
Loss Of Function ◽  
Wild Type ◽  
Limited Effect ◽  
Signal Sequences ◽  
Biochemical Basis ◽  
Mutant Strains ◽  
The Stability ◽  
Synthetic Phenotype

Protein export to the bacterial periplasm is achieved by SecYEG, an inner membrane heterotrimer. SecY and SecE are encoded by essential genes, while SecG is not essential for growth under standard laboratory conditions. Using a quantitative and sensitive export assay, we show that SecG plays a critical role for the residual export mediated by mutant signal sequences; the magnitude of this effect is not proportional to the strength of the export defect. In contrast, export mediated by wild-type signal sequences is only barely retarded in the absence of SecG. When probed with mutant signal sequences,secGloss of function mutations display a phenotype opposite to that ofprlAmutations insecY. The analysis ofsecGandprlAsingle and double mutant strains shows that the increased export conferred by severalprlAalleles is enhanced in the absence of SecG. Several combinations ofprlAalleles with asecGdeletion cannot be easily constructed. This synthetic phenotype is conditional, indicating that cells can adapt to the presence of both alleles. The biochemical basis of this phenomenon is linked to the stability of the SecYE dimer in solubilized membranes. WithprlAalleles that can be normally introduced in asecGdeletion strain, SecG has only a limited effect on the stability of the SecYE dimer. With the otherprlAalleles, the SecYE dimer can often be detected only in the presence of SecG. A possible role for the maintenance of SecG during evolution is proposed.

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