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 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 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.

scholarly journals The carboxy terminal coiled-coil modulates Orai1 internalization during meiosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rawad Hodeify ◽  
Maya Dib ◽  
Ethel Alcantara-Adap ◽  
Raphael Courjaret ◽  
Nancy Nader ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Coiled Coil ◽  
Structural Motif ◽  
Deletion Mutants ◽  
Binding Motif ◽  
Terminal Sequence ◽  
Molecular Determinant ◽  
C Terminus ◽  
The Stability ◽  
Carboxy Terminal

AbstractRegulation of Ca2+ signaling is critical for the progression of cell division, especially during meiosis to prepare the egg for fertilization. The primary Ca2+ influx pathway in oocytes is Store-Operated Ca2+ Entry (SOCE). SOCE is tightly regulated during meiosis, including internalization of the SOCE channel, Orai1. Orai1 is a four-pass membrane protein with cytosolic N- and C-termini. Orai1 internalization requires a caveolin binding motif (CBM) in the N-terminus as well as the C-terminal cytosolic domain. However, the molecular determinant for Orai1 endocytosis in the C-terminus are not known. Here we show that the Orai1 C-terminus modulates Orai1 endocytosis during meiosis through a structural motif that is based on the strength of the C-terminal intersubunit coiled coil (CC) domains. Deletion mutants show that a minimal C-terminal sequence after transmembrane domain 4 (residues 260–275) supports Orai1 internalization. We refer to this region as the C-terminus Internalization Handle (CIH). Access to CIH however is dependent on the strength of the intersubunit CC. Mutants that increase the stability of the coiled coil prevent internalization independent of specific mutation. We further used human and Xenopus Orai isoforms with different propensity to form C-terminal CC and show a strong correlation between the strength of the CC and Orai internalization. Furthermore, Orai1 internalization does not depend on clathrin, flotillin or PIP2. Collectively these results argue that Orai1 internalization requires both the N-terminal CBM and C-terminal CIH where access to CIH is controlled by the strength of intersubunit C-terminal CC.

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 Evidence that the stalk of Drosophila kinesin heavy chain is an alpha-helical coiled coil.

1992 ◽  
Vol 116 (4) ◽  
pp. 957-965 ◽  
Author(s):  
M de Cuevas ◽  
T Tao ◽  
L S Goldstein
Keyword(s):  
Circular Dichroism ◽  
Heavy Chain ◽  
Coiled Coil ◽  
Chain Gene ◽  
Tail Domain ◽  
Major Transitions ◽  
Amino Terminal ◽  
Kinesin Heavy Chain ◽  
Stalk Domain ◽  
Circular Dichroïsm

Kinesin is a mechanochemical enzyme composed of three distinct domains: a globular head domain, a rodlike stalk domain, and a small globular tail domain. The stalk domain has sequence features characteristic of alpha-helical coiled coils. To gain insight into the structure of the kinesin stalk, we expressed it from a segment of the Drosophila melanogaster kinesin heavy chain gene and purified it from Escherichia coli. When observed by EM, this protein formed a rodlike structure 40-55 nm long that was occasionally bent at a hingelike region near the middle of the molecule. An additional EM study and a chemical cross-linking study showed that this protein forms a parallel dimer and that the two chains are in register. Finally, using circular dichroism spectroscopy, we showed that this protein is approximately 55-60% alpha-helical in physiological aqueous solution at 25 degrees C, and approximately 85-90% alpha-helical at 4 degrees C. From these results, we conclude that the stalk of kinesin heavy chain forms an alpha-helical coiled coil structure. The temperature dependence of the circular dichroism signal has two major transitions, at 25-30 degrees C and at 45-50 degrees C, which suggests that a portion of the alpha-helical structure in the stalk is less stable than the rest. By producing the amino-terminal (coil 1) and carboxy-terminal (coil 2) halves of the stalk separately in E. coli, we showed that the region that melts below 30 degrees C lies within coil 1, while the majority of coil 2 melts above 45 degrees C. We suggest that this difference in stability may play a role in the force-generating mechanism or regulation of kinesin.

Immunocytochemical methodology for the localization of neuronal antigens at the ultrastructural level

1990 ◽  
Vol 48 (3) ◽  
pp. 876-877
Author(s):  
Jorge Pecci Saavedra ◽  
Mark Connaughton ◽  
Juan José López ◽  
Alicia Brusco
Keyword(s):  
Spinal Cord ◽  
Substantia Nigra ◽  
Polyclonal Antibodies ◽  
Ultrastructural Level ◽  
Point Of View ◽  
Nerve Tissue ◽  
Tissue Fixation ◽  
Optimal Conditions ◽  
Interpeduncular Nucleus ◽  
Technical Point

The use of antibodies as labels for the localization of specific molecules in the nervous systan has been extensively applied in recent years. Both monoand polyclonal antibodies or antisera have been employed. The knowledge of the organization of neuronal connectivities, gliovascular relationships, glioneuronal relationships and other features of nerve tissue has greatly increased.A number of areas of the nervous systan have been analyzed in our laboratory, including the nuclei of the raphe system, the reticular formation, interpeduncular nucleus, substantia nigra, caudate nucleus, putamen, pallidum, spinal cord, pineal gland and others.From a technical point of view, a number of variables needed to be taken into account in order to obtain reliable and reproducible results. The design of the optimal conditions of tissue fixation, embedding, sectioning, dilution of antibodies, and adaptation of Sternberger PAP technique were sane of the parameters taken into account to optimize the results. It is critical that each step of the technique be defined for each particular case.

Interaction Between Mutations in the suppressor of Hairy wing and modifier of mdg4 Genes of Drosophila melunogaster Affecting the Phenotype of gypsy-Induced Mutations

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 425-436 ◽  
Author(s):  
Pavel Georgiev ◽  
Marina Kozycina
Keyword(s):  
Mutagenic Effect ◽  
Induced Mutations ◽  
Binding Region ◽  
Direct Inhibition ◽  
Amino Terminal ◽  
Acidic Domain ◽  
Complete Inactivation ◽  
Insulation Effect ◽  
Gypsy Retrotransposon ◽  
Carboxy Terminal

Abstract The suppressor of Hairy-wing [su(Hw)] protein mediates the mutagenic effect of the gypsy retrotransposon by repressing the function of transcriptional enhancers located distally from the promoter with respect to the position of the su(Hw)-binding region. Mutations in a second gene, modifier of mdg4, also affect the gypsy-induced phenotype. Two major effects of the mod(mdg4)lul mutation can be distinguished: the interference with insulation by the su(Hw)-binding region and direct inhibition of gene expression that is not dependent on the su(Hw)-binding region position. The mod(mdg4)lul mutation partially suppresses ct6, scD1 and Hw1 mutations, possibly by interfering with the insulation effect of the su(Hw)-binding region. An example of the second effect of mod(mdg4)lul is a complete inactivation of yellow expression in combination with the y  2 allele. Phenotypic analyses of flies with combinations of mod(mdg4)lul and different su(Hw) mutations, or with constructions carrying deletions of the acidic domains of the su(Hw) protein, suggest that the carboxy-terminal acidic domain is important for direct inhibition of yellow transcription in bristles, while the amino-terminal acidic domain is more essential for insulation.

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.

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