scholarly journals The NUF1 gene encodes an essential coiled-coil related protein that is a potential component of the yeast nucleoskeleton.

1992 ◽  
Vol 116 (6) ◽  
pp. 1319-1332 ◽  
Author(s):  
C Mirzayan ◽  
C S Copeland ◽  
M Snyder
Keyword(s):  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Coiled Coil ◽  
Immunoblot Analysis ◽  
Expression Library ◽  
Amino Terminal ◽  
Eukaryotic Nucleus ◽  
Nonionic Detergent ◽  
Potential Component ◽  
Carboxy Terminal

In an attempt to identify structural components of the yeast nucleus, subcellular fractions of yeast nuclei were prepared and used as immunogens to generate complex polyclonal antibodies. One such serum was used to screen a yeast genomic lambda gt11 expression library. A clone encoding a gene called NUF1 (for nuclear filament-related) was identified and extensively characterized. Antibodies to NUF1 fusion proteins were generated, and affinity-purified antibodies were used for immunoblot analysis and indirect immunofluorescence localization. The NUF1 protein is 110 kD in molecular mass and localizes to the yeast nucleus in small granular patches. Intranuclear staining is present in cells at all stages of the cell cycle. The NUF1 protein of yeast is tightly associated with the nucleus; it was not removed by extraction of nuclei with nonionic detergent or salt, or treatment with RNAse and DNAse. Sequence analysis of the NUF1 gene predicts a protein 945 amino acids in length that contains three domains: a large 627 residue central domain predicted to form a coiled-coil structure flanked by nonhelical amino-terminal and carboxy-terminal regions. Disruption of the NUF1 gene indicates that it is necessary for yeast cell growth. These results indicate that NUF1 encodes an essential coiled-coil protein within the yeast nucleus; we speculate that NUF1 is a component of the yeast nucleoskeleton. In addition, immunofluorescence results indicate that mammalian cells contain a NUF1-related nuclear protein. These data in conjunction with those in the accompanying manuscript (Yang et al., 1992) lead to the hypothesis that an internal coiled-coil filamentous system may be a general structural component of the eukaryotic nucleus.

scholarly journals Human Tripartite Motif 5α Domains Responsible for Retrovirus Restriction Activity and Specificity

2005 ◽  
Vol 79 (14) ◽  
pp. 8969-8978 ◽  
Author(s):  
David Perez-Caballero ◽  
Theodora Hatziioannou ◽  
Annie Yang ◽  
Simone Cowan ◽  
Paul D. Bieniasz
Keyword(s):  
Mammalian Cells ◽  
Leukemia Virus ◽  
Coiled Coil ◽  
Spry Domain ◽  
Amino Terminal ◽  
Tripartite Motif ◽  
Additional Domain ◽  
Carboxy Terminal ◽  
Necessary And Sufficient ◽  
Hiv 1

ABSTRACT The tripartite motif 5α protein (TRIM5α) is one of several factors expressed by mammalian cells that inhibit retrovirus replication. Human TRIM5α (huTRIM5α) inhibits infection by N-tropic murine leukemia virus (N-MLV) but is inactive against human immunodeficiency virus type 1 (HIV-1). However, we show that replacement of a small segment in the carboxy-terminal B30.2/SPRY domain of huTRIM5α with its rhesus macaque counterpart (rhTRIM5α) endows it with the ability to potently inhibit HIV-1 infection. The B30.2/SPRY domain and an additional domain in huTRIM5α, comprising the amino-terminal RING and B-box components of the TRIM motif, are required for N-MLV restriction activity, while the intervening coiled-coil domain is necessary and sufficient for huTRIM5α multimerization. Truncated huTRIM5α proteins that lack either or both the N-terminal RING/B-Box or the C-terminal B30.2/SPRY domain form heteromultimers with full-length huTRIM5α and are dominant inhibitors of its N-MLV restricting activity, suggesting that homomultimerization of intact huTRIM5α monomers is necessary for N-MLV restriction. However, localization in large cytoplasmic bodies is not required for inhibition of N-MLV by huTRIM5α or for inhibition of HIV-1 by chimeric or rhTRIM5α.

scholarly journals Properties of the desmin tail domain: studies using synthetic peptides and antipeptide antibodies.

1990 ◽  
Vol 111 (5) ◽  
pp. 2063-2075 ◽  
Author(s):  
L Birkenberger ◽  
W Ip
Keyword(s):  
Polyclonal Antibodies ◽  
Coiled Coil ◽  
Ultrastructural Level ◽  
Structural Motif ◽  
Label Free ◽  
Tail Domain ◽  
Amino Terminal ◽  
Filament Structure ◽  
Carboxy Terminal ◽  
High Degree

Intermediate filament (IF) proteins have a common structural motif consisting of an alpha-helical rod domain flanked by non-alpha-helical amino-terminal head and carboxy-terminal tail domains. Coiled-coil interaction between neighboring rod domains is though to generate the backbone of the 10-nm filament. There must also be other interactions between subunits to bring them into alignment and to effect elongation of the filament, but these are poorly understood. To examine the involvement of the tail domain in filament structure and stabilization, we have studied the interaction between a synthetic peptide corresponding to residues 442-450 of avian desmin, and authentic desmin protein. The potential importance of this region lies in its hydrophilic nature and its high degree of homology among the Type III IF proteins and cytokeratins 8 and 18. The peptide, D442-450, binds to a 27-residue region between lys-436 and leu-463, the carboxy terminus. The presence of the peptide during assembly causes the filaments to appear much more loosely packed than normal desmin IF. We have also generated polyclonal antibodies against this peptide and attempted to localize this portion of the tailpiece along desmin IFs by immunological procedures. By immunoblotting, we found that anti-D442-450 antibodies recognize desmin and only those proteolytic fragments that contain the tailpiece. In contrast, the antibodies do not label any structure in adult gizzard smooth muscle and skeletal muscle myofibrils in immunofluorescence experiments during which conventional antidesmin antibodies do. At the ultrastructural level, anti-D442-450 antibodies label free desmin tetramers but not desmin IFs. These results show that, as part of an assembled IF, the epitope of anti-D442-450 is inaccessible to the antibodies, and suggest that either the tailpiece of an IF protein may not be entirely peripheral to the filament backbone, or the interaction between end domains during assembly masks this particular region of the IF molecule.

scholarly journals The git5 Gβ and git11 Gγ Form an Atypical Gβγ Dimer Acting in the Fission Yeast Glucose/cAMP Pathway

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1159-1168 ◽  
Author(s):  
Sheila Landry ◽  
Charles S Hoffman
Keyword(s):  
Fission Yeast ◽  
Mammalian Cells ◽  
Glucose Repression ◽  
Coiled Coil ◽  
Carboxy Terminus ◽  
Amino Terminal ◽  
Camp Pathway ◽  
Coiled Coil Domain ◽  
Mutational Activation ◽  
Two Hybrid

AbstractFission yeast adenylate cyclase, like mammalian adenylate cyclases, is regulated by a heterotrimeric G protein. The gpa2 Gα and git5 Gβ are both required for glucose-triggered cAMP signaling. The git5 Gβ is a unique member of the Gβ family in that it lacks an amino-terminal coiled-coil domain shown to be essential for mammalian Gβ folding and interaction with Gγ subunits. Using a git5 bait in a two-hybrid screen, we identified the git11 Gγ gene. Co-immunoprecipitation studies confirm the composition of this Gβγ dimer. Cells deleted for git11 are defective in glucose repression of both fbp1 transcription and sexual development, resembling cells lacking either the gpa2 Gα or the git5 Gβ. Overexpression of the gpa2 Gα partially suppresses loss of either the git5 Gβ or the git11 Gγ, while mutational activation of the Gα fully suppresses loss of either Gβ or Gγ. Deletion of gpa2 (Gα), git5 (Gβ), or git11 (Gγ) confer quantitatively distinct effects on fbp1 repression, indicating that the gpa2 Gα subunit remains partially active in the absence of the Gβγ dimer and that the git5 Gβ subunit remains partially active in the absence of the git11 Gγ subunit. The addition of the CAAX box from the git11 Gγ to the carboxy-terminus of the git5 Gβ partially suppresses the loss of the Gγ. Thus the Gγ in this system is presumably required for localization of the Gβγ dimer but not for folding of the Gβ subunit. In mammalian cells, the essential roles of the Gβ amino-terminal coiled-coil domains and Gγ partners in Gβ folding may therefore reflect a mechanism used by cells that express multiple forms of both Gβ and Gγ subunits to regulate the composition and activity of its G proteins.

scholarly journals The Catenin p120ctnInteracts with Kaiso, a Novel BTB/POZ Domain Zinc Finger Transcription Factor

1999 ◽  
Vol 19 (5) ◽  
pp. 3614-3623 ◽  
Author(s):  
Juliet M. Daniel ◽  
Albert B. Reynolds
Keyword(s):  
Transcription Factor ◽  
Monoclonal Antibodies ◽  
Cell Adhesion ◽  
Mammalian Cells ◽  
Yeast Two Hybrid ◽  
Amino Terminal ◽  
Juxtamembrane Region ◽  
Carboxy Terminal ◽  
Two Hybrid ◽  
E Cadherin

ABSTRACT p120 ctn is an Armadillo repeat domain protein with structural similarity to the cell adhesion cofactors β-catenin and plakoglobin. All three proteins interact directly with the cytoplasmic domain of the transmembrane cell adhesion molecule E-cadherin; β-catenin and plakoglobin bind a carboxy-terminal region in a mutually exclusive manner, while p120 binds the juxtamembrane region. Unlike β-catenin and plakoglobin, p120 does not interact with α-catenin, the tumor suppressor adenomatous polyposis coli (APC), or the transcription factor Lef-1, suggesting that it has unique binding partners and plays a distinct role in the cadherin-catenin complex. Using p120 as bait, we conducted a yeast two-hybrid screen and identified a novel transcription factor which we named Kaiso. Kaiso’s deduced amino acid sequence revealed an amino-terminal BTB/POZ protein-protein interaction domain and three carboxy-terminal zinc fingers of the C2H2 DNA-binding type. Kaiso thus belongs to a rapidly growing family of POZ-ZF transcription factors that include the Drosophila developmental regulators Tramtrak and Bric à brac, and the human oncoproteins BCL-6 and PLZF, which are causally linked to non-Hodgkins’ lymphoma and acute promyelocytic leukemia, respectively. Monoclonal antibodies to Kaiso were generated and used to immunolocalize the protein and confirm the specificity of the p120-Kaiso interaction in mammalian cells. Kaiso specifically coprecipitated with a variety of p120-specific monoclonal antibodies but not with antibodies to α- or β-catenin, E-cadherin, or APC. Like other POZ-ZF proteins, Kaiso localized to the nucleus and was associated with specific nuclear dots. Yeast two-hybrid interaction assays mapped the binding domains to Arm repeats 1 to 7 of p120 and the carboxy-terminal 200 amino acids of Kaiso. In addition, Kaiso homodimerized via its POZ domain but it did not heterodimerize with BCL-6, which heterodimerizes with PLZF. The involvement of POZ-ZF proteins in development and cancer makes Kaiso an interesting candidate for a downstream effector of cadherin and/or p120 signaling.

Centrosomal components immunologically related to tektins from ciliary and flagellar microtubules

1994 ◽  
Vol 107 (8) ◽  
pp. 2095-2105 ◽  
Author(s):  
W. Steffen ◽  
E.A. Fajer ◽  
R.W. Linck
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Immunoblot Analysis ◽  
Surf Clam ◽  
Alpha Tubulin ◽  
Sea Urchin Sperm

Centrosomes are critical for the nucleation and organization of the microtubule cytoskeleton during both interphase and cell division. Using antibodies raised against sea urchin sperm flagellar microtubule proteins, we characterize here the presence and behavior of certain components associated with centrosomes of the surf clam Spisula solidissima and cultured mammalian cells. A Sarkosyl detergent-resistant fraction of axonemal microtubules was isolated from sea urchin sperm flagella and used to produce monoclonal antibodies, 16 of which were specific- or cross-specific for the major polypeptides associated with this microtubule fraction: tektins A, B and C, acetylated alpha-tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelectric focussing/SDS polyacrylamide gel electrophoresis the tektins separate into several polypeptide spots. Identical spots were recognized by monoclonal and polyclonal antibodies against a given tektin, indicating that the different polypeptide spots are isoforms or modified versions of the same protein. Four independently derived monoclonal anti-tektins were found to stain centrosomes of S. solidissima oocytes and CHO and HeLa cells, by immunofluorescence microscopy. In particular, the centrosome staining of one monoclonal antibody specific for tektin B (tekB3) was cell-cycle-dependent for CHO cells, i.e. staining was observed only from early prometaphase until late anaphase. By immuno-electron microscopy tekB3 specifically labeled material surrounding the centrosome, whereas a polyclonal anti-tektin B recognized centrioles as well as the centrosomal material throughout the cell cycle. Finally, by immunoblot analysis tekB3 stained polypeptides of 48–50 kDa in isolated spindles and centrosomes from CHO cells.

scholarly journals Novel 45-Kilodalton Leptospiral Protein That Is Processed to a 31-Kilodalton Growth-Phase-Regulated Peripheral Membrane Protein

2002 ◽  
Vol 70 (1) ◽  
pp. 323-334 ◽  
Author(s):  
James Matsunaga ◽  
Tracy A. Young ◽  
Jeanne K. Barnett ◽  
Dean Barnett ◽  
Carole A. Bolin ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Peptide Mapping ◽  
Immunoblot Analysis ◽  
Mammalian Host ◽  
Protein Antigens ◽  
Expression Library ◽  
Genomic Expression ◽  
Amino Terminal ◽  
Peripheral Membrane Protein ◽  
Low Passage

ABSTRACT Leptospiral protein antigens are of interest as potential virulence factors and as candidate serodiagnostic and immunoprotective reagents. We identified leptospiral protein antigens by screening a genomic expression library with serum from a rabbit hyperimmunized with formalin-killed, virulent Leptospira kirschneri serovar grippotyphosa. Genes expressing known outer membrane lipoproteins LipL32 and LipL41, the heat shock protein GroEL, and the α, β, and β′ subunits of RNA polymerase were isolated from the library. In addition, a new leptospiral gene that in Escherichia coli expressed a 45-kDa antigen with an amino-terminal signal peptide followed by the spirochetal lipobox Val−4-Phe−3-Asn−2-Ala−1↓Cys+1 was isolated. We designated this putative lipoprotein LipL45. Immunoblot analysis of a panel of Leptospira strains probed with LipL45 antiserum demonstrated that many low-passage strains expressed LipL45. In contrast, LipL45 was not detected in high-passage, culture-attenuated strains, suggesting that LipL45 is a virulence-associated protein. In addition, all leptospiral strains tested, irrespective of culture passage, expressed a 31-kDa antigen that was recognized by LipL45 antiserum. Southern blot and peptide mapping studies indicated that this 31-kDa antigen was derived from the carboxy terminus of LipL45; therefore, it was designated P31LipL45. Membrane fractionation studies demonstrated that P31LipL45 is a peripheral membrane protein. Finally, we found that P31LipL45 levels increased as Leptospira entered the stationary phase, indicating that P31LipL45 levels were regulated. Hamsters infected with L. kirschneri formed an antibody response to LipL45, indicating that LipL45 was expressed during infection. Furthermore, the immunohistochemistry of kidneys from infected hamsters indicated that LipL45 was expressed by L. kirschneri that colonized the renal tubule. These observations suggest that expression of LipL45 responds to environmental cues, including those encountered during infection of a mammalian host.

scholarly journals NuMA: an unusually long coiled-coil related protein in the mammalian nucleus.

1992 ◽  
Vol 116 (6) ◽  
pp. 1303-1317 ◽  
Author(s):  
C H Yang ◽  
E J Lambie ◽  
M Snyder
Keyword(s):  
Mammalian Cells ◽  
Coiled Coil ◽  
Nuclear Lamina ◽  
Early Prophase ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Mitotic Apparatus ◽  
Reading Frame ◽  
Nuclear Lamins

A bank of 892 autoimmune sera was screened by indirect immunofluorescence on mammalian cells. Six sera were identified that recognize an antigen(s) with a cell cycle-dependent localization pattern. In interphase cells, the antibodies stained the nucleus and in mitotic cells the spindle apparatus was recognized. Immunological criteria indicate that the antigen recognized by at least one of these sera corresponds to a previously identified protein called the nuclear mitotic apparatus protein (NuMA). A cDNA which partially encodes NuMA was cloned from a lambda gt11 human placental cDNA expression library, and overlapping cDNA clones that encode the entire gene were isolated. DNA sequence analysis of the clones has identified a long open reading frame capable of encoding a protein of 238 kD. Analysis of the predicted protein sequence suggests that NuMA contains an unusually large central alpha-helical domain of 1,485 amino acids flanked by nonhelical terminal domains. The central domain is similar to coiled-coil regions in structural proteins such as myosin heavy chains, cytokeratins, and nuclear lamins which are capable of forming filaments. Double immunofluorescence experiments performed with anti-NuMA and antilamin antibodies indicate that NuMA dissociates from condensing chromosomes during early prophase, before the complete disintegration of the nuclear lamina. As mitosis progresses, NuMA reassociates with telophase chromosomes very early during nuclear reformation, before substantial accumulation of lamins on chromosomal surfaces is evident. These results indicate that the NuMA proteins may be a structural component of the nucleus and may be involved in the early steps of nuclear reformation during telophase.

scholarly journals Formation of Membrane-bound Ring Complexes by Prohibitins in Mitochondria

2005 ◽  
Vol 16 (1) ◽  
pp. 248-259 ◽  
Author(s):  
Takashi Tatsuta ◽  
Kirstin Model ◽  
Thomas Langer
Keyword(s):  
Cell Cycle Progression ◽  
Coiled Coil ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
High Molecular Weight Complex ◽  
Amino Terminal ◽  
Mitochondrial Inner Membrane ◽  
Membrane Bound ◽  
Ring Complexes ◽  
Carboxy Terminal

Prohibitins comprise a remarkably conserved protein family in eukaryotic cells with proposed functions in cell cycle progression, senescence, apoptosis, and the regulation of mitochondrial activities. Two prohibitin homologues, Phb1 and Phb2, assemble into a high molecular weight complex of ∼1.2 MDa in the mitochondrial inner membrane, but a nuclear localization of Phb1 and Phb2 also has been reported. Here, we have analyzed the biogenesis and structure of the prohibitin complex in Saccharomyces cerevisiae. Both Phb1 and Phb2 subunits are targeted to mitochondria by unconventional noncleavable targeting sequences at their amino terminal end. Membrane insertion involves binding of newly imported Phb1 to Tim8/13 complexes in the intermembrane space and is mediated by the TIM23-translocase. Assembly occurs via intermediate-sized complexes of ∼120 kDa containing both Phb1 and Phb2. Conserved carboxy-terminal coiled-coil regions in both subunits mediate the formation of large assemblies in the inner membrane. Single particle electron microscopy of purified prohibitin complexes identifies diverse ring-shaped structures with outer dimensions of ∼270 × 200 Å. Implications of these findings for proposed cellular activities of prohibitins are discussed.

scholarly journals SMC1: an essential yeast gene encoding a putative head-rod-tail protein is required for nuclear division and defines a new ubiquitous protein family.

1993 ◽  
Vol 123 (6) ◽  
pp. 1635-1648 ◽  
Author(s):  
A V Strunnikov ◽  
V L Larionov ◽  
D Koshland
Keyword(s):  
Nuclear Division ◽  
Coiled Coil ◽  
Eukaryotic Cell ◽  
Fundamental Aspect ◽  
Nucleoside Triphosphate ◽  
Gene Encoding ◽  
Amino Terminal ◽  
New Family ◽  
Chromosome Iii ◽  
Carboxy Terminal

The smc1-1 mutant was identified initially as a mutant of Saccharomyces cerevisiae that had an elevated rate of minichromosome nondisjunction. We have cloned the wild-type SMC1 gene. The sequence of the SMC1 gene predicts that its product (Smc1p) is a 141-kD protein, and antibodies against Smc1 protein detect a protein with mobility of 165 kD. Analysis of the primary and putative secondary structure of Smc1p suggests that it contains two central coiled-coil regions flanked by an amino-terminal nucleoside triphosphate (NTP)-binding head and a conserved carboxy-terminal tail. These analyses also indicate that Smc1p is an evolutionary conserved protein and is a member of a new family of proteins ubiquitous among prokaryotes and eukaryotes. The SMC1 gene is essential for viability. Several phenotypic characteristics of the mutant alleles of smc1 gene indicate that its product is involved in some aspects of nuclear metabolism, most likely in chromosome segregation. The smc1-1 and smc1-2 mutants have a dramatic increase in mitotic loss of a chromosome fragment and chromosome III, respectively, but have no increase in mitotic recombination. Depletion of SMC1 function in the ts mutant, smc1-2, causes a dramatic mitosis-related lethality. Smc1p-depleted cells have a defect in nuclear division as evidenced by the absence of anaphase cells. This phenotype of the smc1-2 mutant is not RAD9 dependent. Based upon the facts that Smc1p is a member of a ubiquitous family, and it is essential for yeast nuclear division, we propose that Smc1p and Smc1p-like proteins function in a fundamental aspect of prokaryotic and eukaryotic cell division.

scholarly journals Network of protein interactions within the Drosophila inner kinetochore

Open Biology ◽  
2016 ◽  
Vol 6 (2) ◽  
pp. 150238 ◽  
Author(s):  
Magdalena M. Richter ◽  
Jaroslaw Poznanski ◽  
Anna Zdziarska ◽  
Mariusz Czarnocki-Cieciura ◽  
Zoltan Lipinszki ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mutational Analysis ◽  
Coiled Coil ◽  
Amino Terminal ◽  
Carboxy Terminal Domain ◽  
Binding Surface ◽  
Hydrogen Deuterium ◽  
Carboxy Terminal ◽  
And Function ◽  
Kinetochore Function

The kinetochore provides a physical connection between microtubules and the centromeric regions of chromosomes that is critical for their equitable segregation. The trimeric Mis12 sub-complex of the Drosophila kinetochore binds to the mitotic centromere using CENP-C as a platform. However, knowledge of the precise connections between Mis12 complex components and CENP-C has remained elusive despite the fundamental importance of this part of the cell division machinery. Here, we employ hydrogen–deuterium exchange coupled with mass spectrometry to reveal that Mis12 and Nnf1 form a dimer maintained by interacting coiled-coil (CC) domains within the carboxy-terminal parts of both proteins. Adjacent to these interacting CCs is a carboxy-terminal domain that also interacts with Nsl1. The amino-terminal parts of Mis12 and Nnf1 form a CENP-C-binding surface, which docks the complex and thus the entire kinetochore to mitotic centromeres. Mutational analysis confirms these precise interactions are critical for both structure and function of the complex. Thus, we conclude the organization of the Mis12–Nnf1 dimer confers upon the Mis12 complex a bipolar, elongated structure that is critical for kinetochore function.

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