The single CH domain of calponin is neither sufficient nor necessary for F-actin binding

1998 ◽  
Vol 111 (13) ◽  
pp. 1813-1821 ◽  
Author(s):  
M. Gimona ◽  
R. Mital
Keyword(s):  
Binding Site ◽  
Tandem Repeats ◽  
Actin Binding ◽  
Carboxyl Terminus ◽  
Amino Terminus ◽  
Binding Motif ◽  
Amino Terminal ◽  
Nonmuscle Cells ◽  
Carboxy Terminal

Calponins have been implicated in the regulation of actomyosin interactions in smooth muscle cells, cytoskeletal organisation in nonmuscle cells, and the control of neurite outgrowth. Domains homologous to the amino-terminal region of calponin have been identified in a variety of actin cross-linking proteins and signal transduction molecules, and by inference these ‘calponin homology (CH) domains’ have been assumed to participate in actin binding. We here report on the actin binding activities of the subdomains of the calponin molecule. All three mammalian isoforms of calponin (basic h1, neutral h2 and acidic) possess a single CH domain at their amino terminus as well as three tandem repeats proximal to the carboxyl terminus. Calponin h2 differs, however, from h1 in lacking a consensus actin-binding motif in the region 142–163, between the CH domain and the tandem repeats, which in h1 calponin can be chemically cross-linked to actin. Despite the absence of this consensus actin-binding motif, recombinant full-length h2 calponin co-sediments in vitro with F-actin, suggesting the presence of another binding site in the molecule. It could be shown that this binding site resides in the C-terminal tandem repeats and not in the CH domain. Thus, constructs of h2 calponin bearing partial or complete deletions of the triple repeated sequences failed to co-localise with actin stress fibres despite the presence of a CH domain. Deletion of the acidic carboxyl terminus, beyond the repeats, increased actin binding, suggesting that the carboxy-terminal tail may modulate actin association. Results obtained from transient transfections of amino- and carboxy-terminal truncations in h1 calponin were consistent with the established location of the actin binding motif outside and carboxy-terminal to the CH domain, and confirm that the presence of a single CH domain alone is neither sufficient nor necessary to mediate actin binding. Instead, the carboxy-terminal tandem repeats of h1 and h2 calponin are shown to harbour a second, independent actin binding motif.

scholarly journals Multiple genes coding for precursors of rhodotorucine A, a farnesyl peptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides.

1989 ◽  
Vol 9 (8) ◽  
pp. 3491-3498 ◽  
Author(s):  
R Akada ◽  
K Minomi ◽  
J Kai ◽  
I Yamashita ◽  
T Miyakawa ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Rhodosporidium Toruloides ◽  
Basidiomycetous Yeast ◽  
Peptide Sequence ◽  
In Vitro Translation ◽  
Mating Pheromone ◽  
S1 Nuclease ◽  
Amino Terminal ◽  
Carboxy Terminal

Haploid cells of mating type A of the basidiomycetous yeast Rhodosporidium toruloides secrete a mating pheromone, rhodotorucine A, which is an undecapeptide containing S-farnesyl cysteine at its carboxy terminus. To analyze the processing and secretion pathway of rhodotorucine A, we isolated both genomic and complementary DNAs encoding the peptide moiety. We identified three distinct genes, RHA1, RHA2, and RHA3, encoding four, five, and three copies of the pheromone peptide, respectively. Complementary DNA clones were classified into two types. One type was homologous to RHA1, and the other type was homologous to RHA2. Transcription start sites were identified by primer extension and S1 nuclease protection, from which the site of the initiator methionine was verified. A primary precursor of rhodotorucine A was detected as a 7-kilodalton protein by immunoprecipitation of in vitro translation products. On the basis of these results, we propose similar three-precursor structures of rhodotorucine A, each containing the amino-terminal peptide sequence Met-Val-Ala. The precursors contain three, four, or five tandem repeats of the pheromone peptide, each separated by a spacer peptide, Thr-Val-Ser(Ala)-Lys, and each precursor has the carboxy-terminal sequence Thr-Val-Ala. This structure suggests that primary precursors of rhodotorucine A do not contain canonical signal sequences.

Multiple genes coding for precursors of rhodotorucine A, a farnesyl peptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides

1989 ◽  
Vol 9 (8) ◽  
pp. 3491-3498
Author(s):  
R Akada ◽  
K Minomi ◽  
J Kai ◽  
I Yamashita ◽  
T Miyakawa ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Rhodosporidium Toruloides ◽  
Basidiomycetous Yeast ◽  
Peptide Sequence ◽  
In Vitro Translation ◽  
Mating Pheromone ◽  
S1 Nuclease ◽  
Amino Terminal ◽  
Carboxy Terminal

Haploid cells of mating type A of the basidiomycetous yeast Rhodosporidium toruloides secrete a mating pheromone, rhodotorucine A, which is an undecapeptide containing S-farnesyl cysteine at its carboxy terminus. To analyze the processing and secretion pathway of rhodotorucine A, we isolated both genomic and complementary DNAs encoding the peptide moiety. We identified three distinct genes, RHA1, RHA2, and RHA3, encoding four, five, and three copies of the pheromone peptide, respectively. Complementary DNA clones were classified into two types. One type was homologous to RHA1, and the other type was homologous to RHA2. Transcription start sites were identified by primer extension and S1 nuclease protection, from which the site of the initiator methionine was verified. A primary precursor of rhodotorucine A was detected as a 7-kilodalton protein by immunoprecipitation of in vitro translation products. On the basis of these results, we propose similar three-precursor structures of rhodotorucine A, each containing the amino-terminal peptide sequence Met-Val-Ala. The precursors contain three, four, or five tandem repeats of the pheromone peptide, each separated by a spacer peptide, Thr-Val-Ser(Ala)-Lys, and each precursor has the carboxy-terminal sequence Thr-Val-Ala. This structure suggests that primary precursors of rhodotorucine A do not contain canonical signal sequences.

scholarly journals Residues near the Amino Terminus of Rns Are Essential for Positive Autoregulation and DNA Binding

2008 ◽  
Vol 190 (7) ◽  
pp. 2279-2285 ◽  
Author(s):  
Georgeta N. Basturea ◽  
Maria D. Bodero ◽  
Mario E. Moreno ◽  
George P. Munson
Keyword(s):  
Dna Binding ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain ◽  
Regulated Promoters

ABSTRACT Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.

scholarly journals The carboxyl terminus of phage HK022 Nun includes a novel zinc-binding motif and a tryptophan required for transcription termination

2000 ◽  
Vol 14 (6) ◽  
pp. 731-739
Author(s):  
Randolph S. Watnick ◽  
Stephanie Chiyoko Herring ◽  
Arthur G. Palmer ◽  
Max E. Gottesman
Keyword(s):  
Rna Polymerase ◽  
Rna Binding ◽  
Transcription Termination ◽  
Tryptophan Residue ◽  
Binding Motif ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

The amino-terminal arginine-rich motif of the phage HK022 Nun protein binds phage λ nascent mRNA transcripts while the carboxy-terminal domain binds RNA polymerase and arrests transcription. The role of specific residues in the carboxy-terminal domain in transcription termination were investigated by mutagenesis, in vitro and in vivo functional assays, and NMR spectroscopy. Coordination of zinc to three histidine residues in the carboxy-terminus inhibited RNA binding by the amino-terminal domain; however, only two of these histidines were required for transcription arrest. These results suggest that additional zinc-coordinating residues are supplied by RNA polymerase in the context of the Nun–RNA polymerase complex. Substitution of the penultimate carboxy-terminal tryptophan residue with alanine or leucine blocks transcription arrest, whereas a tyrosine substitution is innocuous. Wild-type Nun fails to arrest transcription on single-stranded templates. These results suggest that Nun inhibition of transcription elongation is due in part to interactions between the carboxy-terminal tryptophan of Nun and double-stranded DNA, possibly by intercalation. A model for the termination activity of Nun is developed on the basis of these data.

scholarly journals Effects of point mutations in the cytidine deaminase domains of APOBEC3B on replication and hypermutation of hepatitis B virus in vitro

2007 ◽  
Vol 88 (12) ◽  
pp. 3270-3274 ◽  
Author(s):  
Marianne Bonvin ◽  
Jobst Greeve
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Point Mutations ◽  
Alternative Mechanism ◽  
Amino Terminal ◽  
Hbv Replication ◽  
B Virus ◽  
Carboxy Terminal ◽  
Deaminase Domain

APOBEC3 cytidine deaminases hypermutate hepatitis B virus (HBV) and inhibit its replication in vitro. Whether this inhibition is due to the generation of hypermutations or to an alternative mechanism is controversial. A series of APOBEC3B (A3B) point mutants was analysed in vitro for hypermutational activity on HBV DNA and for inhibitory effects on HBV replication. Point mutations inactivating the carboxy-terminal deaminase domain abolished the hypermutational activity and reduced the inhibitory activity on HBV replication to approximately 40 %. In contrast, the point mutation H66R, inactivating the amino-terminal deaminase domain, did not affect hypermutations, but reduced the inhibition activity to 63 %, whilst the mutant C97S had no effect in either assay. Thus, only the carboxy-terminal deaminase domain of A3B catalyses cytidine deaminations leading to HBV hypermutations, but induction of hypermutations is not sufficient for full inhibition of HBV replication, for which both domains of A3B must be intact.

Cell transformation by avian defective leukaemia viruses

1980 ◽  
Vol 210 (1180) ◽  
pp. 397-409 ◽  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Cell Transformation ◽  
Nucleic Acid Hybridization ◽  
Phenotypic Analysis ◽  
Amino Terminal ◽  
Cell Specificity ◽  
Core Proteins ◽  
Carboxy Terminal

A comparative study of seven independently isolated defective leukaemia viruses has been carried out. Phenotypic analysis of the chicken bone marrow cells transformed in vitro allowed the separation of these seven viruses into three groups based on the differentiation phenotype of the transformed cell. Nucleic acid hybridization studies revealed that these seven viruses had acquired cellular sequences. Interestingly, these studies also showed that the viruses within the same biological grouping had acquired related sequences. This indicates that viruses that have acquired the same or similar cellular sequences have very similar oncogenic capabilities. Analysis of proteins expressed in cells transformed by these viruses demonstrated that the cellular sequences were usually inserted within the gene for the viral core proteins, gag . Therefore the cellular sequences are expressed as a gag -related fusion protein which has an amino-terminal region derived from the gag gene and a carboxy-terminal half derived from the cellular sequences. Two exceptions to this are discussed. The general conclusion from these studies is that defective leukaemia viruses transform cells by virtue of acquired host cellular sequences. The ability of these viruses to transform cells and the target cell specificity of the transformation depends on these cellular sequences.

Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association

1992 ◽  
Vol 12 (3) ◽  
pp. 1087-1095
Author(s):  
M Werner ◽  
S Hermann-Le Denmat ◽  
I Treich ◽  
A Sentenac ◽  
P Thuriaux
Keyword(s):  
Rna Polymerase ◽  
Zinc Binding ◽  
Enzyme Function ◽  
Directed Mutagenesis ◽  
Binding Motif ◽  
Zinc Ions ◽  
Amino Terminal ◽  
Zinc Binding Domain ◽  
Zinc Binding Motif

The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif CX2CX6-12CXGHXGX24-37CX2C, present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.

Mitotic arrest caused by the amino terminus of Xenopus cyclin B2

1993 ◽  
Vol 13 (3) ◽  
pp. 1480-1488
Author(s):  
H M van der Velden ◽  
M J Lohka
Keyword(s):  
Sea Urchin ◽  
Kinase Activity ◽  
Histone H1 ◽  
Amino Terminus ◽  
Protein Kinase Activity ◽  
Truncated Protein ◽  
Amino Terminal ◽  
Egg Extracts ◽  
The Stability

Progression through mitosis requires the inactivation of the protein kinase activity of the p34cdc2-cyclin complex by a mechanism involving the degradation of cyclin. We have examined the stability in Xenopus egg extracts of radiolabeled Xenopus or sea urchin B-type cyclins synthesized in reticulocyte lysates. Xenopus cyclin B2 and sea urchin cyclin B were stable in metaphase extracts from unfertilized eggs but were specifically degraded following addition of Ca2+ to the extracts. The degradation of either cyclin was inhibited by the addition of an excess of unlabeled Xenopus cyclin B2 but not by the addition of a number of control proteins. A truncated protein containing only the amino terminus of Xenopus cyclin B2, including sequences known to be essential for cyclin degradation in other species, also inhibited cyclin degradation, even though the truncated protein was stable in extracts following Ca2+ addition. The addition of the truncated protein did not stimulate histone H1 kinase activity in extracts but prevented the loss of H1 kinase activity that normally follows Ca2+ addition to metaphase extracts. When the amino-terminal fragment was added to extracts capable of several cell cycles in vitro, progression through the first mitosis was inhibited and elevated histone H1 kinase activity was maintained. These results indicate that although the amino terminus of cyclin does not contain all of the information necessary for cyclin destruction, it is capable of interacting with components of the cyclin destruction pathway and thereby preventing the degradation of full-length cyclins.

scholarly journals The carboxy-terminal domain of Gs alpha is necessary for anchorage of the activated form in the plasma membrane.

1990 ◽  
Vol 111 (4) ◽  
pp. 1427-1435 ◽  
Author(s):  
Y Audigier ◽  
L Journot ◽  
C Pantaloni ◽  
J Bockaert
Keyword(s):  
Adenylate Cyclase ◽  
Binding Proteins ◽  
Alpha Subunit ◽  
Amino Terminus ◽  
Gtp Binding Proteins ◽  
Amino Terminal ◽  
Alpha Subunits ◽  
Gtp Binding ◽  
Carboxy Terminal ◽  
Gs Alpha

GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the alpha beta gamma-type. In the activated state, the alpha subunit dissociates from the beta gamma complex but remains anchored in the membrane. The alpha subunits of several GTP-binding proteins, such as Go and Gi, are myristoylated at the amino terminus (Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Proc. Natl. Acad. Sci. USA. 84:7493-7497). This hydrophobic modification is crucial for their membrane attachment. The absence of fatty acid on the alpha subunit of Gs (Gs alpha), the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gs alpha, we used a reconstitution model in which posttranslational addition of in vitro-translated Gs alpha to cyc- membranes (obtained from a mutant of S49 cell line which does not express Gs alpha) restores the coupling between the beta-adrenergic receptor and adenylate cyclase. The consequence of deletions generated by proteolytic removal of amino acid sequences or introduced by genetic removal of coding sequences was determined by analyzing membrane association of the proteolyzed or mutated alpha chains. Proteolytic removal of a 9-kD amino-terminal domain or genetic deletion of 28 amino-terminal amino acids did not modify the anchorage of Gs alpha whereas proteolytic removal of a 1-kD carboxyterminal domain abolished membrane interaction. Thus, in contrast to the myristoylated alpha subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gs alpha are required for association of this protein with the membrane.

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