scholarly journals Characterization of human torsinA and its dystonia-associated mutant form

2003 ◽  
Vol 374 (1) ◽  
pp. 117-122 ◽  
Author(s):  
Zhonghua LIU ◽  
Anna ZOLKIEWSKA ◽  
Michal ZOLKIEWSKI
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Signal Sequence ◽  
Eukaryotic Cells ◽  
Membrane Association ◽  
S2 Cells ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Terminal Amino ◽  
Membrane Anchoring

Deletion of a single glutamate in torsinA correlates with early-onset dystonia, the most severe form of a neurological disorder characterized by uncontrollable muscle contractions. TorsinA is targeted to the ER (endoplasmic reticulum) in eukaryotic cells. We investigated the processing and membrane association of torsinA and the dystonia-associated Glu-deletion mutant (torsinAΔE). We found that the signal sequence of torsinA (residues 1–20 from the 40 amino-acid long N-terminal hydrophobic region) is cleaved in Drosophila S2 cells, as shown by the N-terminal sequencing after partial protein purification. TorsinA is not secreted from S2 cells. Consistently, sodium carbonate extraction and Triton X-114 treatment showed that torsinA is associated with the ER membrane in CHO (Chinese-hamster ovary) cells. In contrast, a variant of torsinA that contains the native signal sequence without the hydrophobic region Ile24–Pro40 does not associate with the membranes in CHO cells, and a truncated torsinA without the 40 N-terminal amino acids is secreted in the S2 culture. Thus the 20-amino-acid-long hydrophobic segment in torsinA, which remains at the N-terminus after signal-peptide cleavage, is responsible for the membrane anchoring of torsinA. TorsinAΔE showed similar cleavage of the 20 N-terminal amino acids and membrane association properties similar to wild-type torsinA but, unlike the wild-type, torsinAΔE was not secreted in the S2 culture even after deletion of the membrane-anchoring segment. This indicates that the dystonia-associated mutation produces a structurally distinct, possibly misfolded, form of torsinA, which cannot be properly processed in the secretory pathway of eukaryotic cells.

The N-terminal amino-latch region of Hlg2 component of staphylococcal bi-component γ-haemolysin is dispensable for prestem release to form β-barrel pores

2020 ◽  
Vol 168 (4) ◽  
pp. 349-354
Author(s):  
Kein Takeda ◽  
Yoshikazu Tanaka ◽  
Jun Kaneko
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Human Erythrocyte ◽  
Haemolytic Activity ◽  
Wild Type ◽  
Amino Acid Deletion ◽  
Recent Proposal ◽  
Terminal Amino ◽  
Delayed Haemolysis ◽  
Component Protein

Abstract The contribution of N-terminal regions of staphylococcal bi-component γ-haemolysin toxin components to haemolytic activity towards human erythrocyte cells was investigated in this study. A deletion construct of N-terminal amino acids 1–10 of Hlg2 (Hlg2 ΔN10), which is the S-component protein of γ-haemolysin, had little effect on its haemolytic activity, whereas N-terminal 1–11 amino acid deletion (Hlg2 ΔN11) significantly delayed haemolysis. Moreover, a deletion of N-terminal amino acids 1–17 of LukF, which is the F-component protein of γ-haemolysin, increased its haemolytic activity in combination with either the wild-type or Hlg2 ΔN10. Unlike the N-terminal amino-latch region of staphylococcal α-haemolysin, which is a single component β-barrel pore-forming toxin, the N-terminal regions present in γ-haemolysin components are dispensable for the haemolytic activity of the bi-component toxin. These results strengthen our recent proposal that staphylococcal bi-component γ-haemolysin toxin uses an N-terminal amino-latch independent molecular switch for prestem release during the formation of β-barrel pores.

Mutational analysis of the variant surface glycoprotein GPI-anchor signal sequence inTrypanosoma brucei

2002 ◽  
Vol 115 (4) ◽  
pp. 805-816 ◽  
Author(s):  
Ulrike Böhme ◽  
George A. M. Cross
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Surface ◽  
Signal Sequence ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Gpi Anchor ◽  
Signal Sequences ◽  
Hydrophobic Region ◽  
High Conservation

The variant surface glycoproteins (VSG) of Trypanosoma brucei are anchored to the cell surface via a glycosylphosphatidylinositol (GPI) anchor. All GPI-anchored proteins are synthesized with a C-terminal signal sequence,which is replaced by a GPI-anchor in a rapid post-translational transamidation reaction. VSG GPI signal sequences are extraordinarily conserved. They contain either 23 or 17 amino acids, a difference that distinguishes the two major VSG classes, and consist of a spacer sequence followed by a more hydrophobic region. The ω amino acid, to which GPI is transferred, is either Ser,Asp or Asn, the ω+2 amino acid is always Ser, and the ω+7 amino acid is almost always Lys. In order to determine whether this high conservation is necessary for GPI anchoring, we introduced several mutations into the signal peptide. Surprisingly, changing the most conserved amino acids, at positions ω+1, ω+2 and ω+7, had no detectable effect on the efficiency of GPI-anchoring or on protein abundance. Several more extensive changes also had no discernable impact on GPI-anchoring. Deleting the entire 23 amino-acid signal sequence or the 15 amino-acid hydrophobic region generated proteins that were not anchored. Instead of being secreted, these truncated proteins accumulated in the endoplasmic reticulum prior to lysosomal degradation. Replacing the GPI signal sequence with a proven cell-surface membrane-spanning domain reduced expression by about 99%and resulted not in cell surface expression but in accumulation close to the flagellar pocket and in non-lysosomal compartments. These results indicate that the high conservation of the VSG GPI signal sequence is not necessary for efficient expression and GPI attachment. Instead, the GPI anchor is essential for surface expression of VSG. However, because the VSG is a major virulence factor, it is possible that small changes in the efficiency of GPI anchoring,undetectable in our experiments, might have influenced the evolution of VSG GPI signal sequences.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽  
1991 ◽  
Vol 34 (4) ◽  
pp. 644-651 ◽  
Author(s):  
Kenneth Koo ◽  
W. Dorsey Stuart
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Neurospora Crassa ◽  
Rna Binding ◽  
Signal Sequence ◽  
Average Length ◽  
Open Reading Frame ◽  
Mrna Transcript ◽  
S1 Nuclease ◽  
Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

scholarly journals SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (5) ◽  
pp. 2154-2164 ◽  
Author(s):  
D J DeMarini ◽  
M Winey ◽  
D Ursic ◽  
F Webb ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Gene Product ◽  
Signal Sequence ◽  
Null Alleles ◽  
Nucleoside Triphosphate ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

SOME ASPECTS OF THE STRUCTURE OF HEMOGLOBIN

1964 ◽  
Vol 42 (6) ◽  
pp. 755-762 ◽  
Author(s):  
David B. Smith
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Partial Amino Acid Sequence ◽  
Heme Pocket ◽  
Terminal Amino ◽  
Polypeptide Chains ◽  
Kinetics Of ◽  
Β Chain

An outline of present ideas concerning the arrangement, folding, and chemistry of the polypeptide chains of hemoglobin is given with some references to present know ledge of myoglobin.New material includes a partial amino acid sequence of the β-chain of horse hemoglobin, details concerning the amino acids lining the heme pocket of horse hemoglobin, and the effects of carboxypeptidases A and B on horse oxy- and horse deoxy-hemoglobin. The kinetics of the latter reactions are not simple. The C-terminal amino acids are released more rapidly from the oxygenated form.

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.

Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate

2001 ◽  
Vol 281 (4) ◽  
pp. G1034-G1043 ◽  
Author(s):  
Kousei Ito ◽  
Hiroshi Suzuki ◽  
Yuichi Sugiyama
Keyword(s):  
Amino Acids ◽  
Multidrug Resistance ◽  
Amino Acid ◽  
Membrane Vesicles ◽  
Single Amino Acid ◽  
Sf9 Cells ◽  
Transport Activity ◽  
Wild Type ◽  
Cationic Amino Acids ◽  
The Difference

Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.

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