The Carboxyl-terminal Region of EBP50 Binds to a Site in the Amino-terminal Domain of Ezrin That Is Masked in the Dormant Molecule

We have examined transfected cells by immunofluorescence microscopy to determine the signals and structural features required for the targeting of integral membrane proteins to the inner nuclear membrane. Lamin B receptor (LBR) is a resident protein of the nuclear envelope inner membrane that has a nucleoplasmic, amino-terminal domain and a carboxyl-terminal domain with eight putative transmembrane segments. The amino-terminal domain of LBR can target both a cytosolic protein to the nucleus and a type II integral protein to the inner nuclear membrane. Neither a nuclear localization signal (NLS) of a soluble protein, nor full-length histone H1, can target an integral protein to the inner nuclear membrane although they can target cytosolic proteins to the nucleus. The addition of an NLS to a protein normally located in the inner nuclear membrane, however, does not inhibit its targeting. When the amino-terminal domain of LBR is increased in size from approximately 22.5 to approximately 70 kD, the chimeric protein cannot reach the inner nuclear membrane. The carboxyl-terminal domain of LBR, separated from the amino-terminal domain, also concentrates in the inner nuclear membrane, demonstrating two nonoverlapping targeting signals in this protein. Signals and structural features required for the inner nuclear membrane targeting of proteins are distinct from those involved in targeting soluble polypeptides to the nucleoplasm. The structure of the nucleocytoplasmic domain of an inner nuclear membrane protein also influences targeting, possibly because of size constraints dictated by the lateral channels of the nuclear pore complexes.

Download Full-text

Ty3 Capsid Mutations Reveal Early and Late Functions of the Amino-Terminal Domain

Journal of Virology ◽

10.1128/jvi.02207-06 ◽

2007 ◽

Vol 81 (13) ◽

pp. 6957-6972 ◽

Cited By ~ 25

Author(s):

Liza S. Z. Larsen ◽

Min Zhang ◽

Nadejda Beliakova-Bethell ◽

Virginia Bilanchone ◽

Anne Lamsa ◽

...

Keyword(s):

Tertiary Structure ◽

Helical Structure ◽

Spherical Particles ◽

P Bodies ◽

Amino Terminal ◽

Terminal Domain ◽

Fluorescence And Electron Microscopy ◽

Carboxyl Terminal ◽

Rna Protection ◽

Amino Terminal Domain

ABSTRACT The Ty3 retrotransposon assembles into 50-nm virus-like particles that occur in large intracellular clusters in the case of wild-type (wt) Ty3. Within these particles, maturation of the Gag3 and Gag3-Pol3 polyproteins by Ty3 protease produces the structural proteins capsid (CA), spacer, and nucleocapsid. Secondary and tertiary structure predictions showed that, like retroviral CA, Ty3 CA contains a large amount of helical structure arranged in amino-terminal and carboxyl-terminal bundles. Twenty-six mutants in which alanines were substituted for native residues were used to study CA subdomain functions. Transposition was measured, and particle morphogenesis and localization were characterized by analysis of protein processing, cDNA production, genomic RNA protection, and sedimentation and by fluorescence and electron microscopy. These measures defined five groups of mutants. Proteins from each group could be sedimented in a large complex. Mutations in the amino-terminal domain reduced the formation of fluorescent Ty3 protein foci. In at least one major homology region mutant, Ty3 protein concentrated in foci but no wt clusters of particles were observed. One mutation in the carboxyl-terminal domain shifted assembly from spherical particles to long filaments. Two mutants formed foci separate from P bodies, the proposed sites of assembly, and formed defective particles. P-body association was therefore found to be not necessary for assembly but correlated with the production of functional particles. One mutation in the amino terminus blocked transposition after cDNA synthesis. Our data suggest that Ty3 proteins are concentrated first, assembly associated with P bodies occurs, and particle morphogenesis concludes with a post-reverse transcription, CA-dependent step. Particle formation was generally resistant to localized substitutions, possibly indicating that multiple domains are involved.

Download Full-text

E2 polypeptides encoded by bovine papillomavirus type 1 form dimers through the common carboxyl-terminal domain: transactivation is mediated by the conserved amino-terminal domain.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.86.2.510 ◽

1989 ◽

Vol 86 (2) ◽

pp. 510-514 ◽

Cited By ~ 81

Author(s):

A. A. McBride ◽

J. C. Byrne ◽

P. M. Howley

Keyword(s):

Bovine Papillomavirus ◽

Amino Terminal ◽

Terminal Domain ◽

Carboxyl Terminal Domain ◽

The Common ◽

Carboxyl Terminal ◽

Amino Terminal Domain

Download Full-text

The Amino-terminal Domain of the Vacuolar Proton-translocating ATPase a Subunit Controls Targeting andin VivoDissociation, and the Carboxyl-terminal Domain Affects Coupling of Proton Transport and ATP Hydrolysis

Journal of Biological Chemistry ◽

10.1074/jbc.m108310200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 47411-47420 ◽

Cited By ~ 124

Author(s):

Shoko Kawasaki-Nishi ◽

Katherine Bowers ◽

Tsuyoshi Nishi ◽

Michael Forgac ◽

Tom H. Stevens

Keyword(s):

Proton Transport ◽

Atp Hydrolysis ◽

Amino Terminal ◽

Terminal Domain ◽

Glucose Depletion ◽

Carboxyl Terminal Domain ◽

A Subunit ◽

Carboxyl Terminal ◽

Amino Terminal Domain ◽

Proton Translocating Atpase

The 100-kDa “a” subunit of the vacuolar proton-translocating ATPase (V-ATPase) is encoded by two genes in yeast,VPH1andSTV1. The Vph1p-containing complex localizes to the vacuole, whereas the Stv1p-containing complex resides in some other intracellular compartment, suggesting that the a subunit contains information necessary for the correct targeting of the V-ATPase. We show that Stv1p localizes to a late Golgi compartment at steady state and cycles continuously via a prevacuolar endosome back to the Golgi. V-ATPase complexes containing Vph1p and Stv1p also differ in their assembly properties, coupling of proton transport to ATP hydrolysis, and dissociation in response to glucose depletion. To identify the regions of the a subunit that specify these different properties, chimeras were constructed containing the cytosolic amino-terminal domain of one isoform and the integral membrane, carboxyl-terminal domain from the other isoform. Like the Stv1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Stv1p localized to the Golgi and the complex did not dissociate in response to glucose depletion. Like the Vph1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Vph1p localized to the vacuole and the complex exhibited normal dissociation upon glucose withdrawal. Interestingly, the V-ATPase complex containing the chimera with the carboxyl-terminal domain of Vph1p exhibited a higher coupling of proton transport to ATP hydrolysis than the chimera containing the carboxyl-terminal domain of Stv1p. Our results suggest that whereas targeting andin vivodissociation are controlled by sequences located in the amino-terminal domains of the subunit a isoforms, coupling efficiency is controlled by the carboxyl-terminal region.

Download Full-text

Genomic structure, chromosomal localization, and conserved alternative splice forms of thrombopoietin

Blood ◽

10.1182/blood.v85.4.981.bloodjournal854981 ◽

1995 ◽

Vol 85 (4) ◽

pp. 981-988 ◽

Cited By ~ 89

Author(s):

AL Gurney ◽

WJ Kuang ◽

MH Xie ◽

BE Malloy ◽

DL Eaton ◽

...

Keyword(s):

Genomic Structure ◽

Single Gene ◽

Amino Acid Deletion ◽

Amino Terminal ◽

Terminal Domain ◽

Carboxyl Terminal Domain ◽

Carboxyl Terminal ◽

Amino Terminal Domain ◽

Splice Forms

Thrombopoietin (TPO), the ligand for c-mpl, is a novel cytokine comprising an amino terminal domain with homology to erythropoietin and a glycosylated carboxyl terminal domain that does not bear overall homology to other known proteins. We report the cloning of cDNAs encoding the porcine and murine TPO and the characterization of the human TPO gene. The cDNA for an additional splice form (TPO-2) with a four-amino-acid deletion within the erythropoietin-like domain has been isolated and is conserved between humans, pigs, and mice. Species comparison of TPO shows that the amino terminal erythropoietin-like domain is highly conserved, while the carboxyl terminal domain is less conserved. Recombinant murine TPO and human TPO are each able to activate both the murine and human c-mpl receptors, indicating an absence of strict species specificity. Human TPO is encoded by a single gene consisting of six exons and located on chromosome 3q271–28.

Download Full-text

Molecular Architecture of the Mouse DNA Polymerase α-Primase Complex

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7886 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7886-7896 ◽

Cited By ~ 56

Author(s):

Takeshi Mizuno ◽

Kumiko Yamagishi ◽

Hiroshi Miyazawa ◽

Fumio Hanaoka

Keyword(s):

Dna Polymerase ◽

Polymerase Activity ◽

Terminal Region ◽

Subunit Assembly ◽

Dna Polymerase Α ◽

Amino Terminal ◽

Terminal Domain ◽

Carboxyl Terminal ◽

Dna Primers ◽

Primase Complex

ABSTRACT The DNA polymerase α-primase complex is the only enzyme that provides RNA-DNA primers for chromosomal DNA replication in eukaryotes. Mouse DNA polymerase α has been shown to consist of four subunits, p180, p68, p54, and p46. To characterize the domain structures and subunit requirements for the assembly of the complex, we constructed eukaryotic polycistronic cDNA expression plasmids expressing pairwise the four subunits of DNA polymerase α. In addition, the constructs contained an internal ribosome entry site derived from poliovirus. The constructs were transfected in different combinations with vectors expressing single subunits to allow the simultaneous expression of three or four of the subunits in cultured mammalian cells. We demonstrate that the carboxyl-terminal region of p180 (residues 1235 to 1465) is essential for its interaction with both p68 and p54-p46 by immunohistochemical analysis and coprecipitation studies with antibodies. Mutations in the putative zinc fingers present in the carboxyl terminus of p180 abolished the interaction with p68 completely, although the mutants were still capable of interacting with p54-p46. Furthermore, the amino-terminal region (residues 1 to 329) and the carboxyl-terminal region (residues 1280 to 1465) were revealed to be dispensable for DNA polymerase activity. Thus, we can divide the p180 subunit into three domains. The first is the amino-terminal domain (residues 1 to 329), which is dispensable for both polymerase activity and subunit assembly. The second is the minimal core domain (residues 330 to 1279), required for polymerase activity. The third is the carboxyl-terminal domain (residues 1280 to 1465), which is dispensable for polymerase activity but required for the interaction with the other three subunits. Taken together, these results allow us to propose the first structural model for the DNA polymerase α-primase complex in terms of subunit assembly, domain structure, and stepwise formation at the cellular level.

Download Full-text

The Carboxyl-terminal Extension of Yeast tRNA m5C Methyltransferase Enhances the Catalytic Efficiency of the Amino-terminal Domain

Journal of Biological Chemistry ◽

10.1074/jbc.m703818200 ◽

2007 ◽

Vol 282 (32) ◽

pp. 23663-23671 ◽

Cited By ~ 8

Author(s):

Hélène Walbott ◽

Sylvie Auxilien ◽

Henri Grosjean ◽

Béatrice Golinelli-Pimpaneau

Keyword(s):

Catalytic Efficiency ◽

Amino Terminal ◽

Yeast Trna ◽

Terminal Domain ◽

Carboxyl Terminal ◽

Amino Terminal Domain

Download Full-text

ADA3, a putative transcriptional adaptor, consists of two separable domains and interacts with ADA2 and GCN5 in a trimeric complex.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1203 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1203-1209 ◽

Cited By ~ 104

Author(s):

J Horiuchi ◽

N Silverman ◽

G A Marcus ◽

L Guarente

Keyword(s):

Activation Domains ◽

Amino Terminal ◽

Terminal Domain ◽

Trimeric Complex ◽

Nonoverlapping Domains ◽

Carboxyl Terminal Domain ◽

Carboxyl Terminal ◽

Amino Terminal Domain

Mutations in yeast ADA2, ADA3, and GCN5 weaken the activation potential of a subset of acidic activation domains. In this report, we show that their gene products form a heterotrimeric complex in vitro, with ADA2 as the linchpin holding ADA3 and GCN5 together. Further, activation by LexA-ADA3 fusions in vivo are regulated by the levels of ADA2. Combined with a prior observation that LexA-ADA2 fusions are regulated by the levels of ADA3 (N. Silverman, J. Agapite, and L. Guarente, Proc. Natl. Acad. Sci. USA 91:11665-11668, 1994), this finding suggests that these proteins also form a complex in cells. ADA3 can be separated into two nonoverlapping domains, an amino-terminal domain and a carboxyl-terminal domain, which do not separately complement the slow-growth phenotype or transcriptional defect of a delta ada3 strain but together supply full complementation. The carboxyl-terminal domain of ADA3 alone suffices for heterotrimeric complex formation in vitro and activation of LexA-ADA2 in vivo. We present a model depicting the ADA complex as a coactivator in which the ADA3 amino-terminal domain mediates an interaction between activation domains and the ADA complex.

Download Full-text

Different structures in the amino-terminal domain of the ornithine transcarbamylase leader peptide are involved in mitochondrial import and carboxyl-terminal cleavage.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.85.23.8905 ◽

1988 ◽

Vol 85 (23) ◽

pp. 8905-8909 ◽

Cited By ~ 18

Author(s):

J. P. Kraus ◽

J. Novotny ◽

F. Kalousek ◽

M. Swaroop ◽

L. E. Rosenberg

Keyword(s):

Ornithine Transcarbamylase ◽

Leader Peptide ◽

Mitochondrial Import ◽

Amino Terminal ◽

Terminal Domain ◽

Carboxyl Terminal ◽

Amino Terminal Domain

Download Full-text

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645194 ◽

1990 ◽

Vol 63 (02) ◽

pp. 193-203 ◽

Cited By ~ 9

Author(s):

John R Shainoff ◽

Deborah J Stearns ◽

Patricia M DiBello ◽

Youko Hishikawa-Itoh

Keyword(s):

Peritoneal Macrophages ◽

Affinity Binding ◽

Macrophage Cell ◽

High Affinity Binding ◽

Amino Terminal ◽

High Affinity ◽

Terminal Domain ◽

Weak Binding ◽

Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

Download Full-text