The Acinetobacter baylyi hfq Gene Encodes a Large Protein with an Unusual C Terminus

ABSTRACT In gammaproteobacteria the Hfq protein shows a great variation in size, especially in its C-terminal part. Extremely large Hfq proteins consisting of almost 200 amino acid residues and more are found within the gammaproteobacterial family Moraxellaceae. The difference in size compared to other Hfq proteins is due to a glycine-rich domain near the C-terminal end of the protein. Acinetobacter baylyi, a nonpathogenic soil bacterium and member of the Moraxellaceae encodes a large 174-amino-acid Hfq homologue containing the unique and repetitive amino acid pattern GGGFGGQ within the glycine-rich domain. Despite the presence of the C-terminal extension, A. baylyi Hfq complemented an Escherichia coli hfq mutant in vivo. By using polyclonal anti-Hfq antibodies, we detected the large A. baylyi Hfq that corresponds to its annotated size indicating the expression and stability of the full protein. Deletion of the complete A. baylyi hfq open reading frame resulted in severe reduction of growth. In addition, a deletion or overexpression of Hfq was accompanied by the loss of cell chain assembly. The glycine-rich domain was not responsible for growth and cell phenotypes. hfq gene localization in A. baylyi is strictly conserved within the mutL-miaA-hfq operon, and we show that hfq expression starts within the preceding miaA gene or further upstream.

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Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e07-05-0436 ◽

2007 ◽

Vol 18 (8) ◽

pp. 2893-2903 ◽

Cited By ~ 29

Author(s):

Sarah L. Barker ◽

Linda Lee ◽

B. Daniel Pierce ◽

Lymarie Maldonado-Báez ◽

David G. Drubin ◽

...

Keyword(s):

Late Stage ◽

Actin Polymerization ◽

Fluorescent Protein ◽

Temperature Sensitive ◽

Type I ◽

Reading Frame ◽

Rich Domain ◽

C Terminus

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1ΔPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.

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Polymorphism of the yeast pyruvate carboxylase 2 gene and protein: effects on protein biotinylation

Biochemical Journal ◽

10.1042/bj3120817 ◽

1995 ◽

Vol 312 (3) ◽

pp. 817-825 ◽

Cited By ~ 19

Author(s):

D L Val ◽

A Chapman-Smith ◽

M E Walker ◽

J E Cronan ◽

J C Wallace

Keyword(s):

Amino Acid ◽

Pyruvate Carboxylase ◽

Physical Map ◽

Open Reading Frame ◽

Reading Frame ◽

Biotin Ligase ◽

C Terminus ◽

Single Base Deletion ◽

Domain Peptide

In Saccharomyces cerevisiae there are two isoenzymes of pyruvate carboxylase (Pyc) encoded by separate genes designated PYC1 and PYC2. We report the isolation and sequencing of a PYC2 gene, and the localization of both genes on the physical map of S. cerevisiae. Comparison with the previously reported sequence [Stucka, Dequin, Salmon and Gancedo (1991) Mol. Gen. Genet. 229, 307-315] revealed significant differences within the open reading frame. The most notable difference was near the 3′ end, where we found a single base deletion reducing the open reading frame by 15 bases. We have confirmed the C-terminus of Pyc2 encoded by the gene isolated here by expressing and purifying an 86-amino-acid biotin-domain peptide. In addition, we investigated the effects of the two changes in the Pyc2 biotin domain (K1155R substitution and Q1178P/five-amino-acid extension) on the extent of biotinylation in vivo by Escherichia coli biotin ligase, and compared the biotinylation of peptides containing these changes with that of two different-length Pyc1 biotin-domain peptides. The K1155R substitution had very little effect on biotinylation, but the five-amino-acid C-terminal extension to Pyc2 and the N-terminal extension to Pycl both improved biotinylation in vivo.

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Identification of the gene for disaggregatase fromMethanosarcina mazei

Archaea ◽

10.1155/2008/949458 ◽

2008 ◽

Vol 2 (3) ◽

pp. 185-191 ◽

Cited By ~ 3

Author(s):

Naoki Osumi ◽

Yoshihiro Kakehashi ◽

Shiho Matsumoto ◽

Kazunari Nagaoka ◽

Junichi Sakai ◽

...

Keyword(s):

Amino Acid ◽

Single Cells ◽

Pcr Analysis ◽

Amino Acid Residues ◽

Wall Function ◽

Rt Pcr ◽

Methanosarcina Mazei ◽

C Terminus ◽

First Time

The gene sequences encoding disaggregatase (Dag), the enzyme responsible for dispersion of cell aggregates ofMethanosarcina mazeito single cells, were determined for three strains ofM. mazei(S-6T, LYC and TMA). Thedaggenes of the three strains were 3234 bp in length and had almost the same sequences with 97% amino acid sequence identities. Dag was predicted to comprise 1077 amino acid residues and to have a molecular mass of 120 kDa containing three repeats of the DNRLRE domain in the C terminus, which is specific to the genusMethanosarcinaand may be responsible for structural organization and cell wall function. Recombinant Dag was overexpressed inEscherichia coliand preparations of the expressed protein exhibited enzymatic activity. The RT-PCR analysis showed thatdagwas transcribed to mRNA inM. mazeiLYC and indicated that the gene was expressed in vivo. This is the first time the gene involved in the morphological change ofMethanosarcinaspp. from aggregate to single cells has been identified.

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Separate bisphosphatase domain of chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: the role of the C-terminal tail in modulating enzyme activity

Biochemical Journal ◽

10.1042/bj3280751 ◽

1997 ◽

Vol 328 (3) ◽

pp. 751-756 ◽

Cited By ~ 6

Author(s):

Lin LI ◽

Song LING ◽

Chun-lai WU ◽

Wei-zhe YAO ◽

Gen-jun XU

Keyword(s):

Enzyme Activity ◽

Amino Acid ◽

Substrate Inhibition ◽

Fold Increase ◽

Chicken Liver ◽

Bifunctional Enzyme ◽

Amino Acid Residues ◽

C Terminus ◽

Km Value ◽

The Difference

The separate bisphosphatase domain (amino acid residues 243-468) of the chicken liver bifunctional enzyme 6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase was expressed in Escherichia coli and purified to homogeneity. The fructose-2,6-bisphosphatase activity of the separate domain was 7-fold higher than that of the native bifunctional enzyme, and exhibited substrate inhibition characteristic of the native enzyme. The inhibition of the enzymes by fructose 2,6-bisphosphate could be overcome by Pi, glycerol 3-phosphate and GTP. Deletion of 30 amino acid residues from the C-terminus of the separate domain resulted in around a 5-fold increase in the Vmax of the bisphosphatase. Also, the truncated form was more accessible to chemical modification by diethyl pyrocarbonate and N-ethylmaleimide, suggesting a more open structure than the wild-type form. In addition, the mutation of cysteine-389 to alanine increased bisphosphatase activity by 20% and the Km value for fructose 2,6-bisphosphate by 3-fold, and both the point mutation at cysteine-389 and the deletional mutation led to the predominantly insoluble expression of the enzyme. The results indicated that the C-terminal tail plays a role in modulating the enzyme activity and suggested that the difference in the C-terminal tail sequence is responsible for the difference in activity of the chicken and rat liver fructose-2,6-bisphosphatases. It is postulated that an interaction between the C-terminal tail and the active site might be present.

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Molecular cloning of a cDNA coding for mouse liver xanthine dehydrogenase. Regulation of its transcript by interferons in vivo

Biochemical Journal ◽

10.1042/bj2830863 ◽

1992 ◽

Vol 283 (3) ◽

pp. 863-870 ◽

Cited By ~ 83

Author(s):

M Terao ◽

G Cazzaniga ◽

P Ghezzi ◽

M Bianchi ◽

F Falciani ◽

...

Keyword(s):

Amino Acid ◽

Mouse Liver ◽

De Novo ◽

Xanthine Dehydrogenase ◽

Amino Acid Residues ◽

Cross Hybridization ◽

Reading Frame ◽

Ifn Alpha ◽

High Conservation

The cDNA coding for xanthine dehydrogenase (XD) is isolated from mouse liver mRNA by cross-hybridization with a DNA fragment of the Drosophila melanogaster homologue. Two lambda bacteriophage overlapping clones represent the copy of a 4538-nucleotide-residue-long transcript with an open reading frame of 4005 nucleotide residues, coding for a putative polypeptide of 1335 amino acid residues. Comparison of the deduced amino acid sequence of the mouse XD with those of the Drosophila and the rat homologues shows a high conservation of this protein (55% identity between mouse and Drosophila, and 94% identity between mouse and rat). RNA blotting analysis demonstrates that interferon-alpha (IFN-alpha) and its inducers, i.e. poly(I).poly(C), bacterial lipopolysaccharide (LPS) and tilorone (2,7-bis-[2-(diethylamino)ethoxy]fluoren-9-one), increase the expression of XD mRNA in liver. Poly(I).poly(C) also induces XD mRNA in several other tissues in vivo. Protein synthesis de novo is not required for the elevation of XD mRNA after IFN-alpha treatment, since cycloheximide does not block the induction. The elevation of XD mRNA concentration is relatively fast and precedes the induction of both XD and xanthine oxidase (XO) enzymic activities.

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Isolation and characterization of cDNA for human 120 kDa mitochondrial 2,4-dienoyl-coenzyme A reductase

Biochemical Journal ◽

10.1042/bj3040787 ◽

1994 ◽

Vol 304 (3) ◽

pp. 787-792 ◽

Cited By ~ 26

Author(s):

K T Koivuranta ◽

E H Hakkola ◽

J K Hiltunen

Keyword(s):

Amino Acid ◽

Amino Acid Sequences ◽

Cdna Clones ◽

Human Tissues ◽

Amino Acid Residues ◽

Beta Oxidation ◽

Reading Frame ◽

Isolation And Characterization ◽

The Difference ◽

Coa Reductase

2,4-Dienoyl-CoA reductase (EC 1.3.1.34) participates in beta-oxidation of (poly)unsaturated enoyl-CoAs and it appears in mammalian mitochondria as two isoforms with molecular masses of 120 and 60 kDa [Hakkola and Hiltunen (1993) Eur. J. Biochem. 215, 199-204]. The 120 kDa isomer is a homotetrameric enzyme, and here we report cDNA cloning of its subunit from human. cDNA clones were isolated by reverse transcriptase-PCR from a fibrosarcoma cell line and by screening from a human liver lambda gt11 cDNA library. The 1128 bp clone contained an open reading frame of 1008 bp encoding a polypeptide of 335 amino acid residues with a predicted molecular mass of 36066 Da. This polypeptide represents the immature monomer of the 120 kDa enzyme, and it contains a predicted N-terminal mitochondrial targeting signal. The amino acid (nucleotide) sequence of human 2,4-dienoyl-CoA reductase shows 82.7% (81.7%) similarity (identity) to the corresponding sequence from the rat. Northern-blot analysis gave a single mRNA species of 1.2 kb in several human tissues, the amounts present in the tissues tested ranking as follows: heart approximately liver approximately pancreas > kidney >> skeletal muscle approximately lung. Immunoblotting of human and rat liver samples with an antibody to the subunit of the rat 120 kDa isoform indicates that the mature human enzyme is larger than its counterpart in the rat. The comparison of amino acid sequences for rat and human enzymes proposes that the difference in the size is 10 amino acid residues. The results show that the rat and human reductases are similar in many characteristics and that the reductase is expressed in human tissues capable of beta-oxidation of fatty acids.

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Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19872317 ◽

1987 ◽

Vol 52 (9) ◽

pp. 2317-2325 ◽

Cited By ~ 5

Author(s):

Jan Hlaváček ◽

Jan Pospíšek ◽

Jiřina Slaninová ◽

Walter Y. Chan ◽

Victor J. Hruby

Keyword(s):

Amino Acid ◽

Solid Phase Synthesis ◽

Solid Phase ◽

The Other ◽

Amino Acid Residues ◽

Phase Synthesis ◽

Other Hand ◽

Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

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Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19951229 ◽

1995 ◽

Vol 60 (7) ◽

pp. 1229-1235 ◽

Cited By ~ 2

Author(s):

Ivana Zoulíková ◽

Ivan Svoboda ◽

Jiří Velek ◽

Václav Kašička ◽

Jiřina Slaninová ◽

...

Keyword(s):

Amino Acid ◽

Biological Activities ◽

Residual Activity ◽

Detailed Examination ◽

Amino Acid Residues ◽

Linear Peptide ◽

Zone Electrophoresis ◽

Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

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Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium

Biochemical Journal ◽

10.1042/bj2510691 ◽

1988 ◽

Vol 251 (3) ◽

pp. 691-699 ◽

Cited By ~ 106

Author(s):

R W Olafson ◽

W D McCubbin ◽

C M Kay

Keyword(s):

Amino Acid ◽

Metal Binding ◽

Helical Structure ◽

Basic Amino Acid ◽

Cysteine Residues ◽

Amino Acid Residues ◽

C Terminus ◽

Empirical Relations ◽

Heavy Metal Binding ◽

Synechococcus Sp

Biochemical and physiological studies of Synechococcus cyanobacteria have indicated the presence of a low-Mr heavy-metal-binding protein with marked similarity to eukaryotic metallothioneins (MTs). We report here the characterization of a Synechococcus prokaryotic MT isolated by gel-permeation and reverse-phase chromatography. The large number of variants of this molecule found during chromatographic separation could not be attributed to the presence of major isoproteins as assessed by amino acid analysis and amino acid sequencing of isoforms. Two of the latter were shown to have identical primary structures that differed substantially from the well-described eukaryotic MTs. In addition to six long-chain aliphatic residues, two aromatic residues were found adjacent to one another near the centre of the molecule, making this the most hydrophobic MT to be described. Other unusual features included a pair of histidine residues located in repeating Gly-His-Thr-Gly sequences near the C-terminus and a complete lack of association of hydroxylated residues with cysteine residues, as is commonly found in eukaryotes. Similarly, aside from a single lysine residue, no basic amino acid residues were found adjacent to cysteine residues in the sequence. Most importantly, sequence alignment analyses with mammalian, invertebrate and fungal MT sequences showed no statistically significant homology aside from the presence of Cys-Xaa-Cys structures common to all MTs. On the other hand, like other MTs, the prokaryotic molecule appears to be free of alpha-helical structure but has a considerable amount of beta-structure, as predicted by both c.d. measurements and the Chou & Fasman empirical relations. Considered together, these data suggested that some similarity between the metal-thiolate clusters of the prokaryote and eukaryote MTs may exist.

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Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons—New Insights into the Role of the C-Terminal Region of Tpm3.1

Cells ◽

10.3390/cells10030715 ◽

2021 ◽

Vol 10 (3) ◽

pp. 715

Author(s):

Tamara Tomanić ◽

Claire Martin ◽

Holly Stefen ◽

Esmeralda Parić ◽

Peter Gunning ◽

...

Keyword(s):

Amino Acid ◽

Hippocampal Neurons ◽

Molecular Mechanisms ◽

Growth Cones ◽

Amino Acid Residues ◽

Terminal Amino Acid ◽

C Terminus ◽

Primary Mouse ◽

Terminal Amino

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

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