scholarly journals Targeting of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE

2005 ◽  
Vol 168 (7) ◽  
pp. 1039-1051 ◽  
Author(s):  
Akira Honda ◽  
Omayma S. Al-Awar ◽  
Jesse C. Hay ◽  
Julie G. Donaldson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Membrane Traffic ◽  
Snare Protein ◽  
Wild Type ◽  
Rab Family ◽  
In Cells

Arf and Rab family GTPases regulate membrane traffic in cells, yet little is known about how they are targeted to distinct organelles. To identify sequences in Arf-1 necessary for Golgi targeting, we examined the localization of chimeras between Arf-1 and Arf-6. Here, we identify a 16–amino acid sequence in Arf-1 that specifies Golgi targeting and contains a motif (MXXE) that is important for Arf-1 binding to membrin, an ER-Golgi SNARE protein. The MXXE motif is conserved in all Arfs known to localize to the Golgi and enables Arf-1 to localize to the early Golgi. Arf-1 lacking these 16 aa can still localize to the late Golgi where it displays a more rapid Golgi-cytosol cycle than wild-type Arf-1. These studies suggest that membrin recruits Arf-1 to the early Golgi and reveal distinct kinetic cycles for Arf-1 at early and late Golgi determined by different sets of Arf regulators and effectors.

scholarly journals Identification of shorter length lamin A protein in mouse ear cartilage tissue

2015 ◽  
Vol 20 (2) ◽  
Author(s):  
Ramamoorthy M. Kalidas ◽  
Subramanian Elaiya Raja ◽  
Sivasubramaniam Sudhakar
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Accelerated Aging ◽  
Cartilage Tissue ◽  
Lamin A ◽  
Intermediate Filament Protein ◽  
Mouse Ear ◽  
Ear Cartilage ◽  
Filament Protein ◽  
In Cells

AbstractLamin A is an intermediate filament protein which is cleaved by the enzyme, FACE 1 at VTRSY↓L. The cleavage is the final step in the production of the mature lamin A protein. The mature lamin A protein localizes in the inner membrane of the nucleus. The mutation in the lamin A gene causes many diseases, including accelerated aging. It is known that the protein is not expressed in neuronal cells of the brain. Many splicing variants of the lamin A gene have been reported. In this study, the amino acid sequence VTRSY (a penta-peptide repeat) was found in three different sites of the C-terminal end of the lamin A protein, the protein expressed in cells of ear cartilage tissues is shorter than the protein expressed in cells of the skin tissues. Using two lamin A antibodies, it was found that the amino acid sequence between penta-peptide 2 and 3 is missing in lamin A protein that was expressed in the cells of mouse ear cartilage tissue, besides the RT-PCR data confirmed that the corresponding coding sequence between the penta repeat 2 and 3 is intact. Cleavage may occur at the penta-peptide (VTRSY) at site 3 in the lamin A tail of mouse ear cartilage.

Molecular Characterization of Genes Coding for Wild-Type and Sulfonylurea-Resistant Acetolactate Synthase in the Cyanobacterium Synechococcus PC C7942

1990 ◽  
Vol 45 (5) ◽  
pp. 538-543 ◽  
Author(s):  
D. Friedberg ◽  
J. Seijffers
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Characterization ◽  
Amino Acid Level ◽  
Acetolactate Synthase ◽  
Mutant Gene ◽  
Wild Type ◽  
E Coli

We present here the isolation and molecular characterization of acetolactate synthase (ALS) genes from the cyanobacterium Synechococcus PCC7942 which specify a sulfonylurea-sensitive enzyme and from the sulfonylurea-resistant mutant SM3/20, which specify resistance to sulfonylurea herbicides. The ALS gene was cloned and mapped by complementation of an Escherichia coli ilv auxotroph that requires branched-chain amino acids for growth and lacks ALS activity. The cyanobacterial gene is efficiently expressed in this heterologous host. The ALS gene codes for 612 amino acids and shows high sequence homology (46%) at the amino acid level with ALS III of E. coli and with the tobacco ALS. The resistant phenotype is a consequence of proline to serine substitution in residue 115 of the deduced amino acid sequence. Functional expression of the mutant gene in wild-type Synechococcus and in E. coli confirmed that this amino-acid substitution is responsible for the resistance. Yet the deduced amino-acid sequence as compared with othjer ALS proteins supports the notion that the amino-acid context of the substitution is important for the resistance.

scholarly journals Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

1996 ◽  
Vol 314 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Johanneke L. H. BUSCH ◽  
Jacques L. J. BRETON ◽  
Barry M. BARTLETT ◽  
Richard JAMES ◽  
E. Claude HATCHIKIAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Magnetic Circular Dichroism ◽  
Wild Type ◽  
E Coli ◽  
Excess Iron ◽  
Expression In Escherichia Coli ◽  
Type D

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

Enhanced motility of a Proteus mirabilis strain expressing hybrid FlaAB flagella

Microbiology ◽  
2004 ◽  
Vol 150 (5) ◽  
pp. 1291-1299 ◽  
Author(s):  
Jim Manos ◽  
Elena Artimovich ◽  
Robert Belas
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Proteus Mirabilis ◽  
Motion Tracking ◽  
Antigenic Variation ◽  
Bacterial Species ◽  
Helical Structure ◽  
Swimming Velocity ◽  
Wild Type ◽  
Hybrid Genes

Proteus mirabilis has two tandemly arranged flagellin-encoding genes, flaA and flaB. flaA is transcribed from a σ 28 promoter, while flaB is silent. flaA and flaB can undergo reversible rearrangement to produce a set of hybrid genes referred to as flaAB. Flagellins composed of FlaAB protein have a different amino acid sequence and are antigenically distinct from flagellin composed of FlaA, implicating flagellin gene conversion as a putative virulence mechanism for P. mirabilis. The change in amino acid sequence is also hypothesized to alter the filament helix and, hence, affect the motility of FlaAB-expressing strains. To test this hypothesis, the motility of wild-type P. mirabilis was compared with that of a strain, DF1003, locked into the FlaAB+ hybrid phase, under conditions of altered ionic strength, pH and viscosity. Cell motion tracking analysis showed that DF1003 has wild-type swimming velocity at physiological conditions, but moves significantly faster and travels further compared to the wild-type at NaCl concentrations greater than 170 mM. DF1003 is also significantly faster than the wild-type at pH 5·2, 5·8 and 8·2, and at 5 and 10 % polyvinylpyrrolidone. Measurements of amplitude and wavelength for isolated flagella subjected to pH 5·8 or 425 mM NaCl showed a loss of helical structure in FlaA flagella compared to FlaAB filaments, a feature that could significantly affect motility under these conditions. These results support a hypothesis that FlaAB flagellin imparts a motile advantage to P. mirabilis in conditions that otherwise may impede bacterial movement. In a broader context, flagellar antigenic variation, commonly thought to serve as means to avoid host defences, may also enhance motility in other bacterial species, thus aiding in the adaptation and survival of the cells.

Purification, cloning, and DNA sequence analysis of a chitinase from an overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis

2001 ◽  
Vol 47 (3) ◽  
pp. 179-187 ◽  
Author(s):  
Kuzhandhaivel S Vetrivel ◽  
Shunmugiah K Pandian ◽  
Uma Chaudhary ◽  
Kuppamuthu Dharmalingam
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Catalytic Domain ◽  
Chitinase Gene ◽  
Wild Type ◽  
Terminal Amino Acid ◽  
Streptomyces Peucetius ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using λ DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a λ DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.Key words: chitinase, chitinase purification, Streptomyces peucetius, daunorubicin, chiC.

scholarly journals Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

1999 ◽  
Vol 10 (6) ◽  
pp. 1821-1836 ◽  
Author(s):  
Grigory B. Melikyan ◽  
Sasa Lin ◽  
Michael G. Roth ◽  
Fredric S. Cohen
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Membrane Fusion ◽  
Integral Membrane Protein ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Tm Domain ◽  
Fusion Pores ◽  
Sequence Requirements

The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved.

scholarly journals Interaction between FliE and FlgB, a Proximal Rod Component of the Flagellar Basal Body ofSalmonella

2000 ◽  
Vol 182 (11) ◽  
pp. 3029-3036 ◽  
Author(s):  
Tohru Minamino ◽  
Shigeru Yamaguchi ◽  
Robert M. Macnab
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Basal Body ◽  
Strong Interactions ◽  
Wild Type ◽  
Body Protein ◽  
C Terminus ◽  
Genes Encoding ◽  
And Function ◽  
Better Than

ABSTRACT FliE is a flagellar basal body protein of Salmonellawhose detailed location and function have not been established. A mutant allele of fliE, which caused extremely poor flagellation and swarming, generated extragenic suppressors, all of which mapped to flgB, one of four genes encoding the basal body rod; the fliE flgB pseudorevertants were better flagellated and swarmed better than the fliE parent, especially when the temperature was reduced from 37 to 30°C. Motility of the pseudorevertants in liquid culture was markedly better than motility on swarm plates; we interpret this to mean that reduced flagellation is less deleterious at low viscous loads. Overproduction of the mutant FliE protein improved the motility of the parentalfliE mutant and its pseudorevertants, though not to wild-type levels. Overproduction of suppressor FlgB (but not wild-type FlgB) in the fliE mutant also resulted in improved motility. The second-site FlgB mutation by itself had no phenotype; cells swarmed as well as wild-type cells. When overproduced, wild-type FliE was dominant over FliE-V99G, but the reverse was not true; that is, overproduced FliE-V99G was not negatively dominant over wild-type FliE. We conclude that the mutant protein has reduced probability of assembly but, if assembled, functions relatively well. Export of the flagellar protein FlgD, which is known to be FliE dependent, was severely impaired by the FliE-V99G mutation but was significantly improved in the suppressor strains. The FliE mutation, V99G, was close to the C terminus of the 104-amino-acid sequence; the suppressing mutations in FlgB were all either G119E or G129D, close to the C terminus of its 138-amino-acid sequence. Affinity blotting experiments between FliE as probe and various basal body proteins as targets and vice versa revealed strong interactions between FliE and FlgB; much weaker interactions between FliE and other rod proteins were observed and probably derive from the known similarities among these proteins. We suggest that FliE subunits constitute a junction zone between the MS ring and the rod and also that the proximal rod structure consists of FlgB subunits.

scholarly journals A Ubiquitin-Conjugating Enzyme Is Essential for Developmental Transitions in Dictyostelium

1997 ◽  
Vol 8 (10) ◽  
pp. 1989-2002 ◽  
Author(s):  
Alexandra Clark ◽  
Anson Nomura ◽  
Sudhasri Mohanty ◽  
Richard A. Firtel
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Gene ◽  
Wild Type ◽  
Cell Type ◽  
Protein Ubiquitination ◽  
High Amino Acid ◽  
Cell Type Specific ◽  
Very High

We have identified a developmentally essential gene,UbcB, by insertional mutagenesis. The encoded protein (UBC1) shows very high amino acid sequence identity to ubiquitin-conjugating enzymes from other organisms, suggesting that UBC1 is involved in protein ubiquitination and possibly degradation during Dictyostelium development. Consistent with the homology of the UBC1 protein to UBCs, the developmental pattern of protein ubiquitination is altered in ubcB-null cells.ubcB-null cells are blocked in the ability to properly execute the developmental transition that occurs between the induction of postaggregative gene expression during mound formation and the induction of cell-type differentiation and subsequent morphogenesis.ubcB-null cells plated on agar form mounds with normal kinetics; however, they remain at this stage for ∼10 h before forming multiple tips and fingers that then arrest. Under other conditions, some of the fingers form migrating slugs, but no culmination is observed. In ubcB-null cells, postaggregative gene transcripts accumulate to very high levels and do not decrease significantly with time as they do in wild-type cells. Expression of cell-type-specific genes is very delayed, with the level of prespore-specific gene expression being significantly reduced compared with that in wild-type cells. lacZ reporter studies using developmentally regulated and cell-type-specific promoters suggest that ubcB-null cells show an unusually elevated level of staining of lacZ reporters expressed in anterior-like cells, a regulatory cell population found scattered throughout the aggregate, and reduced staining of a prespore reporter.ubcB-null cells in a chimeric organism containing predominantly wild-type cells are able to undergo terminal differentiation but show altered spatial localization. In contrast, in chimeras containing only a small fraction of wild-type cells, the mature fruiting body is very small and composed almost exclusively of wild-type cells, with the ubcB-null cells being present as a mass of cells located in extreme posterior of the developing organism. The amino acid sequence analysis of the UbcBopen reading frame (ORF) and the analysis of the developmental phenotypes suggest that tip formation and subsequent development requires specific protein ubiquitination, and possibly degradation.

scholarly journals Purification of a heterodimeric Reelin construct to investigate binding stoichiometry

2020 ◽  
Vol 49 (8) ◽  
pp. 773-779 ◽  
Author(s):  
Liam S. Turk ◽  
Daniel Mitchell ◽  
Davide Comoletti
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Receptor Binding ◽  
Direct Evidence ◽  
Synaptic Function ◽  
Wild Type ◽  
Site Mutation ◽  
Binding Interaction ◽  
Receptor Binding Site ◽  
Binding Stoichiometry

AbstractReelin is a secreted glycoprotein that is integral in neocortex development and synaptic function. Reelin exists as a homodimer with two chains linked by a disulfide bond at cysteine 2101, a feature that is vital to the protein’s function. This is highlighted by the fact that only dimeric Reelin can elicit efficient, canonical signaling, even though a mutated (C2101A) monomeric construct of Reelin retains the capacity to bind to its receptors. Receptor clustering has been shown to be important in the signaling pathway, however direct evidence regarding the stoichiometry of Reelin-receptor binding interaction is lacking. Here we describe the construction and purification of a heterodimeric Reelin construct to investigate the stoichiometry of Reelin-receptor binding and how it affects Reelin pathway signaling. We have devised different strategies and have finalized a protocol to produce a heterodimer of Reelin’s central fragment using differential tagging and tandem affinity chromatography, such that chain A is wild type in amino acid sequence whereas chain B includes a receptor binding site mutation (K2467A). We also validate that the heterodimer is capable of binding to the extracellular domain of one of Reelin’s known receptors, calculating the KD of the interaction. This heterodimeric construct will enable us to understand in greater detail the mechanism by which Reelin interacts with its known receptors and initiates pathway signaling.

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