scholarly journals FepA with Globular Domain Deletions Lacks Activity

2002 ◽  
Vol 184 (19) ◽  
pp. 5508-5512 ◽  
Author(s):  
Hema L. Vakharia ◽  
Kathleen Postle
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Globular Domain ◽  
Cross Link ◽  
Amino Terminal ◽  
Dependent Activity ◽  
Colicin B ◽  
Support Activity

ABSTRACT TonB-gated transporters have β-barrels containing an amino-terminal globular domain that occludes the interior of the barrel. Mutations in the globular domain prevent transport of ligands across the outer membrane. Surprisingly, FepA with deletions of the globular domain (amino acids 3 to 150 and 17 to 150) was previously reported to retain significant sensitivity to colicins B and D and to use ferric enterochelin, all in a TonB-dependent fashion. To further understand TonB interaction with the β-barrel, in the present study, proteins with deletions of amino acids 1 to 152, 7 to 152, 20 to 152, and 17 to 150 in fepA were constructed and expressed in a ΔfepA strain. In contrast to previous studies of fepA globular domain deletions, constructs in this study did not retain sensitivity to colicin B and conferred only marginal sensitivity to colicin D. Consistent with these observations, they failed to bind colicin B and detectably cross-link to TonB in vivo. To address this discrepancy, constructs were tested in other strains, one of which (RWB18-60) did support activity of the FepA globular domain deletion proteins constructed in this study. The characteristics of that strain, as well as the strain in which the ΔFhuA globular domain mutants were seen to be active, suggests the hypothesis that interprotein complementation by two individually nonfunctional proteins restores TonB-dependent activity.

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 Assembly of amino-terminally deleted desmin in vimentin-free cells.

1990 ◽  
Vol 111 (5) ◽  
pp. 1971-1985 ◽  
Author(s):  
J M Raats ◽  
F R Pieper ◽  
W T Vree Egberts ◽  
K N Verrijp ◽  
F C Ramaekers ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hela Cells ◽  
Filament Formation ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Double Label ◽  
Amino Terminal Domain ◽  
Mcf 7 ◽  
Vimentin Filaments

To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin-free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.

scholarly journals Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis.

1991 ◽  
Vol 11 (6) ◽  
pp. 2994-3000 ◽  
Author(s):  
K M Yao ◽  
K White
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rna Binding ◽  
Terminal Region ◽  
Drosophila Virilis ◽  
Functional Domain ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

scholarly journals Characterization of Bacteriophage Lambda Excisionase Mutants Defective in DNA Binding

2000 ◽  
Vol 182 (20) ◽  
pp. 5807-5812 ◽  
Author(s):  
Eun Hee Cho ◽  
Renato Alcaraz ◽  
Richard I. Gumport ◽  
Jeffrey F. Gardner
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Glutamic Acid ◽  
Cooperative Interaction ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Factor For Inversion Stimulation ◽  
Α Helix

ABSTRACT The bacteriophage λ excisionase (Xis) is a sequence-specific DNA binding protein required for excisive recombination. Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA. Efficient excision is achieved through a cooperative interaction between Xis and the host-encoded factor for inversion stimulation as well as a cooperative interaction between Xis and integrase. The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28. Several mutants, defective in promoting excision in vivo, were isolated with mutations at positions encoding polar amino acids in the putative helix (T. E. Numrych, R. I. Gumport, and J. F. Gardner, EMBO J. 11:3797–3806, 1992). We substituted alanines for the polar amino acids in this region. Mutant proteins with substitutions for polar amino acids in the amino-terminal region of the putative helix exhibited decreased excision in vivo and were defective in DNA binding. In addition, an alanine substitution at glutamic acid 40 also resulted in altered DNA binding. This indicates that the hydrophilic face of the α-helix and the region containing glutamic acid 40 may form the DNA binding surfaces of the Xis protein.

scholarly journals The C Terminus of Substrates Is Critical but Not Sufficient for Their Degradation by the Pseudomonas aeruginosa CtpA Protease

2020 ◽  
Vol 202 (16) ◽  
Author(s):  
Sammi Chung ◽  
Andrew J. Darwin
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Outer Membrane ◽  
Content Type ◽  
C Terminus ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Carboxyl Terminal

ABSTRACT Bacterial carboxyl-terminal processing proteases (CTPs) are widely conserved and have been linked to important processes, including signal transduction, cell wall metabolism, and virulence. However, the features that target proteins for CTP-dependent cleavage are unclear. Studies of the Escherichia coli CTP Prc suggested that it cleaves proteins with nonpolar and/or structurally unconstrained C termini, but it is not clear if this applies broadly. Pseudomonas aeruginosa has a divergent CTP, CtpA, which is required for virulence. CtpA works in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. In this study, we investigated if the C termini of two nonhomologous CtpA substrates are important for their degradation. We determined that these substrates have extended C termini compared to those of their closest E. coli homologs. Removing 7 amino acids from these extensions was sufficient to reduce their degradation by CtpA both in vivo and in vitro. Degradation of one truncated substrate was restored by adding the C terminus from the other but not by adding an unrelated sequence. However, modification of the C termini of nonsubstrates, by adding the C-terminal amino acids from a substrate, did not cause their degradation by CtpA. Therefore, the C termini of CtpA substrates are required but not sufficient for their efficient degradation. Although C-terminal truncated substrates were protected from degradation, they still associated with the LbcA-CtpA complex in vivo. Therefore, degradation of a protein by CtpA requires a C terminus-independent interaction with the LbcA-CtpA complex, followed by C terminus-dependent degradation, perhaps because CtpA normally initiates cleavage at a C-terminal site. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are found in all three domains of life, but exactly how they work is poorly understood, including how they recognize substrates. Bacterial CTPs have been associated with virulence, including CtpA of Pseudomonas aeruginosa, which works in complex with the outer membrane lipoprotein LbcA to degrade potentially dangerous peptidoglycan hydrolases. We report an important advance by revealing that efficient degradation by CtpA requires at least two separable phenomena and that one of them depends on information encoded in the substrate C terminus. A C terminus-independent association with the LbcA-CtpA complex is followed by C terminus-dependent cleavage by CtpA. Increased understanding of how CTPs target proteins is significant, due to their links to virulence, peptidoglycan remodeling, and other important processes.

The six amino-terminal amino acids of p60src are sufficient to cause myristylation of p21v-ras

1988 ◽  
Vol 8 (9) ◽  
pp. 3960-3963
Author(s):  
J E Buss ◽  
C J Der ◽  
P A Solski
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Myristic Acid ◽  
Amino Terminus ◽  
Directed Mutagenesis ◽  
Ras Protein ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Crucial Part

We have used oligonucleotide-directed mutagenesis to replace the N-terminal amino acids of p21v-ras with residues which mimic the amino terminus of p60v-src. p21v-ras protein possessing only the first five amino acids of p60src was not myristylated, while substitution of residue 6 (serine) produced a protein p21(GSSKS) which incorporated [3H]myristic acid that was stable to hydroxylamine, sensitive to inhibitors of protein synthesis, and found in both the normally nonacylated precursor and mature forms of p21(GSSKS). This defines the minimum framework of the p60v-src myristylation signal (glycine 2 and serine 6) and identifies serine 6 as a crucial part of that signal for myristylation of a protein in vivo.

Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis

1991 ◽  
Vol 11 (6) ◽  
pp. 2994-3000
Author(s):  
K M Yao ◽  
K White
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rna Binding ◽  
Terminal Region ◽  
Drosophila Virilis ◽  
Functional Domain ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

scholarly journals CENP-E Function at Kinetochores Is Essential for Chromosome Alignment

1997 ◽  
Vol 139 (6) ◽  
pp. 1373-1382 ◽  
Author(s):  
B.T. Schaar ◽  
G.K.T. Chan ◽  
P. Maddox ◽  
E.D. Salmon ◽  
T.J. Yen
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Metaphase Plate ◽  
Motor Domain ◽  
Amino Terminal ◽  
Spindle Poles ◽  
Opposite Pole ◽  
Chromosome Alignment ◽  
Combined Data

CENP-E is a kinesin-like protein that binds to kinetochores and may provide functions that are critical for normal chromosome motility during mitosis. To directly test the in vivo function of CENP-E, we microinjected affinity-purified antibodies to block the assembly of CENP-E onto kinetochores and then examined the behavior of these chromosomes. Chromosomes lacking CENP-E at their kinetochores consistently exhibited two types of defects that blocked their alignment at the spindle equator. Chromosomes positioned near a pole remained mono-oriented as they were unable to establish bipolar microtubule connections with the opposite pole. Chromosomes within the spindle established bipolar connections that supported oscillations and normal velocities of kinetochore movement between the poles, but these bipolar connections were defective because they failed to align the chromosomes into a metaphase plate. Overexpression of a mutant that lacked the amino-terminal 803 amino acids of CENP-E was found to saturate limiting binding sites on kinetochores and competitively blocked endogenous CENP-E from assembling onto kinetochores. Chromosomes saturated with the truncated CENP-E mutant were never found to be aligned but accumulated at the poles or were strewn within the spindle as was the case when cells were microinjected with CENP-E antibodies. As the motor domain was contained within the portion of CENP-E that was deleted, the chromosomal defect is likely attributed to the loss of motor function. The combined data show that CENP-E provides kinetochore functions that are essential for monopolar chromosomes to establish bipolar connections and for chromosomes with connections to both spindle poles to align at the spindle equator. Both of these events rely on activities that are provided by CENP-E's motor domain.

scholarly journals Sequences near the Active Site in Chimeric Penicillin Binding Proteins 5 and 6 Affect Uniform Morphology of Escherichia coli

2003 ◽  
Vol 185 (7) ◽  
pp. 2178-2186 ◽  
Author(s):  
Anindya S. Ghosh ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Globular Domain ◽  
Morphological Effect ◽  
E Coli ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Domain I

ABSTRACT Penicillin binding protein (PBP) 5, a dd-carboxypeptidase that removes the terminal d-alanine from peptide side chains of peptidoglycan, plays an important role in creating and maintaining the uniform cell shape of Escherichia coli. PBP 6, a highly similar homologue, cannot substitute for PBP 5 in this respect. Previously, we localized the shape-maintaining characteristics of PBP 5 to the globular domain that contains the active site (domain I), where PBPs 5 and 6 share substantial identity. To identify the specific segment of domain I responsible for shape control, we created a set of hybrids and determined which ones complemented the aberrant morphology of a misshapen PBP mutant, E. coli CS703-1. Fusion proteins were constructed in which 47, 199 and 228 amino-terminal amino acids of one PBP were fused to the corresponding carboxy-terminal amino acids of the other. The morphological phenotype was reversed only by hybrid proteins containing PBP 5 residues 200 to 228, which are located next to the KTG motif of the active site. Because residues 220 to 228 were identical in these proteins, the morphological effect was determined by alterations in amino acids 200 to 219. To confirm the importance of this segment, we constructed mosaic proteins in which these 20 amino acids were grafted from PBP 5 into PBP 6 and vice versa. The PBP 6/5/6 mosaic complemented the aberrant morphology of CS703-1, whereas PBP 5/6/5 did not. Site-directed mutagenesis demonstrated that the Asp218 and Lys219 residues were important for shape maintenance by these mosaic PBPs, but the same mutations in wild-type PBP 5 did not eliminate its shape-promoting activity. Homologous enzymes from five other bacteria also complemented the phenotype of CS703-1. The overall conclusion is that creation of a bacterial cell of regular diameter and uniform contour apparently depends primarily on a slight alteration of the enzymatic activity or substrate accessibility at the active site of E. coli PBP 5.

scholarly journals Activities of the Serratia marcescens Heme Receptor HasR and Isolated Plug and β-Barrel Domains: the β-Barrel Forms a Heme-Specific Channel

2005 ◽  
Vol 187 (13) ◽  
pp. 4637-4645 ◽  
Author(s):  
Sylvie Létoffé ◽  
Karine Wecker ◽  
Muriel Delepierre ◽  
Philippe Delepelaire ◽  
Cécile Wandersman
Keyword(s):  
Serratia Marcescens ◽  
Outer Membrane ◽  
Three Dimensional ◽  
Membrane Receptors ◽  
Outer Membrane Receptor ◽  
Heme Uptake ◽  
Amino Terminal ◽  
Exogenous Compounds

ABSTRACT The Serratia marcescens hemophore-specific outer membrane receptor HasR is a member of the TonB-dependent family of autoregulated receptors. It can transport either heme itself or heme bound to the hemophore HasA. On the basis of sequence and functional similarities with other TonB-dependent outer membrane receptors whose three-dimensional structures have been determined, a HasR structure model was proposed. The mature HasR protein comprises a 99-residue amino-terminal extension necessary for hasR transcription, followed by a plug domain of 139 amino acids and a β-barrel domain inserted in the outer membrane, the lumen of which is closed by the plug. This model was used to generate hasR deletions encoding HasR proteins with the native signal peptides but lacking either the N-terminal regulatory extension or encoding the plug or the β-barrel alone. The protein lacking the N-terminal extension, HasR Δ11-91, was incorporated in the outer membrane and was fully functional for active uptake of free and hemophore-bound heme. The HasR β-barrel, Δ11-192, was also incorporated in the outer membrane and bound the hemophore but expressed no active heme transport properties. The HasR plug remained in the periplasm. Coexpression of the plug and the β-barrel allowed partial plug insertion in the outer membrane, demonstrating that these two HasR domains interact in vivo. The β-barrel and the plug also interact in vitro. Nevertheless, the two domains did not complement each other to reconstitute an active TonB-dependent receptor for free or hemophore-bound heme uptake. Production of the β-barrel alone selectively increased passive diffusion of heme but not of other exogenous compounds. A mutation at histidine 603, which is required for heme uptake through the wild-type receptor, abolished heme diffusion, showing that HasR Δ11-192 forms a specific heme channel.

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