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1.
Chromatin remodeling proteins interact with pericentrin to regulate centrosome integrity 总被引:1,自引:0,他引:1
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Sillibourne JE Delaval B Redick S Sinha M Doxsey SJ 《Molecular biology of the cell》2007,18(9):3667-3680
Pericentrin is an integral centrosomal component that anchors regulatory and structural molecules to centrosomes. In a yeast two-hybrid screen with pericentrin we identified chromodomain helicase DNA-binding protein 4 (CHD4/Mi2beta). CHD4 is part of the multiprotein nucleosome remodeling deacetylase (NuRD) complex. We show that many NuRD components interacted with pericentrin by coimmunoprecipitation and that they localized to centrosomes and midbodies. Overexpression of the pericentrin-binding domain of CHD4 or another family member (CHD3) dissociated pericentrin from centrosomes. Depletion of CHD3, but not CHD4, by RNA interference dissociated pericentrin and gamma-tubulin from centrosomes. Microtubule nucleation/organization, cell morphology, and nuclear centration were disrupted in CHD3-depleted cells. Spindles were disorganized, the majority showing a prometaphase-like configuration. Time-lapse imaging revealed mitotic failure before chromosome segregation and cytokinesis failure. We conclude that pericentrin forms complexes with CHD3 and CHD4, but a distinct CHD3-pericentrin complex is required for centrosomal anchoring of pericentrin/gamma-tubulin and for centrosome integrity. 相似文献
2.
Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time. 相似文献
3.
Chirivino D Del Maestro L Formstecher E Hupé P Raposo G Louvard D Arpin M 《Molecular biology of the cell》2011,22(3):375-385
In the degradative pathway, the progression of cargos through endosomal compartments involves a series of fusion and maturation events. The HOPS (homotypic fusion and protein sorting) complex is part of the machinery that promotes the progression from early to late endosomes and lysosomes by regulating the exchange of small GTPases. We report that an interaction between subunits of the HOPS complex and the ERM (ezrin, radixin, moesin) proteins is required for the delivery of EGF receptor (EGFR) to lysosomes. Inhibiting either ERM proteins or the HOPS complex leads to the accumulation of the EGFR into early endosomes, delaying its degradation. This impairment in EGFR trafficking observed in cells depleted of ERM proteins is due to a delay in the recruitment of Rab7 on endosomes. As a consequence, the maturation of endosomes is perturbed as reflected by an accumulation of hybrid compartments positive for both early and late endosomal markers. Thus, ERM proteins represent novel regulators of the HOPS complex in the early to late endosomal maturation. 相似文献
4.
J L Salisbury A T Baron D E Coling V E Martindale M A Sanders 《Cell motility and the cytoskeleton》1986,6(2):193-197
Affinity-purified antibodies that recognize the 20,000-dalton molecular weight (20 kd) striated flagellar root protein of Tetraselmis striata have been used to identify antigenic homologs in other eucaryotic organisms of diverse evolutionary origins. Among the green algae, Tetraselmis and Chlamydomonas, and their colorless relative, Polytomella, the 20-kd homologs appear associated with basal bodies. This occurs most prominently in the form of flagellar roots of both striated and microtubule subtended types. Among cultured mammalian cells (PtK2 and primary mouse macrophage cell lines), flagellar root protein homologs appear as basal feet, pericentriolar fibrils, and pericentriolar satellites. Mammalian sperm cells also show flagellar root protein homologs associated with their basal bodies. We envisage a functional role for these fibrous calcium-sensitive contractile proteins in altering the orientation of centrioles or basal bodies with their associated MTOCs by responding to topological calcium fluxes. 相似文献
5.
Lethal giant larvae proteins interact with the exocyst complex and are involved in polarized exocytosis
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Zhang X Wang P Gangar A Zhang J Brennwald P TerBush D Guo W 《The Journal of cell biology》2005,170(2):273-283
The tumor suppressor lethal giant larvae (Lgl) plays a critical role in epithelial cell polarization. However, the molecular mechanism by which Lgl carries out its functions is unclear. In this study, we report that the yeast Lgl proteins Sro7p and Sro77p directly interact with Exo84p, which is a component of the exocyst complex that is essential for targeting vesicles to specific sites of the plasma membrane for exocytosis, and that this interaction is important for post-Golgi secretion. Genetic analyses demonstrate a molecular pathway from Rab and Rho GTPases through the exocyst and Lgl to SNAREs, which mediate membrane fusion. We also found that overexpression of Lgl and t-SNARE proteins not only improves exocytosis but also rescues polarity defects in exocyst mutants. We propose that, although Lgl is broadly distributed in the cells, its localized interaction with the exocyst and kinetic activation are important for the establishment and reenforcement of cell polarity. 相似文献
6.
Potential interactions between membrane components of rat brain synaptic vesicles were analyzed by detergent solubilization followed by size fractionation or immunoprecipitation. The behavior of six synaptic vesicle membrane proteins as well as a plasma membrane protein was monitored by Western blotting. Solubilization of synaptic vesicle membranes in CHAPS resulted in the recovery of a large protein complex that included SV2, p65, p38, vesicle-associated membrane protein, and the vacuolar proton pump. Solubilization in octylglucoside resulted in the preservation of interactions between SV2, p38, and rab3A, while solubilization of synaptic vesicles with Triton X-100 resulted in two predominant interactions, one involving p65 and SV2, and the other involving p38 and vesicle-associated membrane protein. The multicomponent complex preserved with CHAPS solubilization was partially reconstituted following octylglucoside solubilization and subsequent dialysis against CHAPS. Reduction of the CHAPS concentration by gel filtration chromatography resulted in increased recovery of the multicomponent complex. Examination of the large complex isolated from CHAPS-solubilized vesicles by negative stain EM revealed structures with multiple globular domains, some of which were specifically labeled with gold-conjugated antibodies directed against p65 and SV2. The protein interactions defined in this report are likely to underlie aspects of neurotransmitter secretion, membrane traffic, and the spatial organization of vesicles within the nerve terminal. 相似文献
7.
Ectromelia virus encodes a novel family of F-box proteins that interact with the SCF complex 总被引:1,自引:0,他引:1
Poxviruses are notorious for encoding multiple proteins that regulate cellular signaling pathways, including the ubiquitin-proteasome system. Bioinformatics indicated that ectromelia virus, the causative agent of lethal mousepox, encoded four proteins, EVM002, EVM005, EVM154, and EVM165, containing putative F-box domains. In contrast to cellular F-box proteins, the ectromelia virus proteins contain C-terminal F-box domains in conjunction with N-terminal ankyrin repeats, a combination that has not been previously reported for cellular proteins. These observations suggested that the ectromelia virus F-box proteins interact with SCF (Skp1, cullin-1, and F-box) ubiquitin ligases. We focused our studies on EVM005, since this protein had only one ortholog in cowpox virus. Using mass spectrometry, we identified cullin-1 as a binding partner for EVM005, and this interaction was confirmed by overexpression of hemagglutinin (HA)-cullin-1. During infection, Flag-EVM005 and HA-cullin-1 colocalized to distinct cellular bodies. Significantly, EVM005 coprecipitated with endogenous Skp1, cullin-1, and Roc1 and associated with conjugated ubiquitin, suggesting that EVM005 interacted with the components of a functional ubiquitin ligase. Interaction of EVM005 with cullin-1 and Skp1 was abolished upon deletion of the F-box, indicating that the F-box played a crucial role in interaction with the SCF complex. Additionally, EVM002 and EVM154 interacted with Skp1 and conjugated ubiquitin, suggesting that ectromelia virus encodes multiple F-box-containing proteins that regulate the SCF complex. Our results indicate that ectromelia virus has evolved multiple proteins that interact with the SCF complex. 相似文献
8.
Masuda HP Ramos GB de Almeida-Engler J Cabral LM Coqueiro VM Macrini CM Ferreira PC Hemerly AS 《FEBS letters》2004,574(1-3):192-202
Eukaryotic DNA replication requires an ordered and regulated machinery to control G1/S transition. The formation of the pre-replicative complex (pre-RC) is a key step involved in licensing DNA for replication. Here, we identify all putative components of the full pre-RC in the genome of the model plant Arabidopsis thaliana. Different from the other eukaryotes, Arabidopsis houses in its genome two putative homologs of ORC1, CDC6 and CDT1. Two mRNA variants of AtORC4 subunit, with different temporal expression patterns, were also identified. Two-hybrid binary interaction assays suggest a primary architectural organization of the Arabidopsis ORC, in which AtORC3 plays a central role in maintaining the complex associations. Expression profiles differ among pre-RC components suggesting the existence of various forms of the complex, possibly playing different roles during development. In addition, the expression of the putative pre-RC genes in non-proliferating plant tissues suggests that they might have roles in processes other than DNA replication licensing. 相似文献
9.
High mobility group proteins HMGD and HMGZ interact genetically with the Brahma chromatin remodeling complex in Drosophila
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Many pleiotropic roles have been ascribed to small abundant HMG-Box (HMGB) proteins in higher eukaryotes but their precise function has remained enigmatic. To investigate their function genetically we have generated a defined deficiency uncovering the functionally redundant genes encoding HMGD and HMGZ, the Drosophila counterparts of HMGB1-3 in mammals. The resulting mutant is a strong hypomorphic allele of HmgD/Z. Surprisingly this allele is viable and exhibits only minor morphological defects even when homozygous. However, this allele interacts strongly with mutants of the Brahma chromatin remodeling complex, while no interaction was observed with mutant alleles of other remodeling complexes. We also observe genetic interactions between the HmgD/Z deficiency and some, but not all, known Brahma targets. These include the homeotic genes Sex combs reduced and Antennapedia, as well as the gene encoding the cell-signaling protein Rhomboid. In contrast to more general structural roles previously suggested for these proteins, we infer that a major function of the abundant HMGB proteins in Drosophila is to participate in Brahma-dependent chromatin remodeling at a specific subset of Brahma-dependent promoters. 相似文献
10.
11.
M F Wiser 《Experimental parasitology》1991,73(4):515-523
Several distinct classes of Plasmodium proteins have been proposed to interact with the submembrane skeleton of the erythrocyte based upon differential solubility and subcellular localization studies. That the parasite affects the erythrocyte membrane by interacting with the submembrane skeleton is an attractive hypothesis since the membrane skeleton likely regulates many aspects of membrane topography and function. The precise interactions between host and parasite proteins at the molecular level and how the parasite proteins are transported to the erythrocyte membrane are not completely understood. Experiments addressing these questions are under way, and such studies will provide valuable information about the host-parasite interface. In addition, the characterization of the interaction of Plasmodium proteins with the host erythrocyte membrane may also provide new insight into the structure and function of the erythrocyte membrane or membranes in general. 相似文献
12.
Previous studies from this laboratory have demonstrated that plasminogen and angiostatin bind to endothelial cell (EC) surface-associated
actin via their kringles in a specific manner. Heat shock proteins (hsps) like hsp 27 are constitutively expressed by vascular
ECs and regulate actin polymerization, cell growth, and migration. Since many hsps have also been found to be highly abundant
on cell surfaces and there is evidence that bacterial surface hsps may interact with human plasminogen, the purpose of this
study was to determine whether human plasminogen and angiostatin would interact with human hsps. ELISAs were developed in
our laboratory to assess these interactions. It was observed that plasminogen bound to hsps 27, 60, and 70. In all cases,
binding was inhibited (85–90%) by excess (50 mM) lysine indicating kringle involvement. Angiostatin predominantly bound to
hsp 27 and to hsp 70 in a concentration- and kringle-dependent manner. As observed previously for actin, there was concentration-dependent
inhibition of angiostatin’s interaction with hsp 27 by plasminogen. In addition, 30-fold molar excess actin inhibited (up
to 50%), the interaction of plasminogen with all hsps. However, 30-fold molar excess actin could only inhibit the interaction
of angiostatin with hsp 27 by 15–20%. Collectively, these data indicate that (i) while plasminogen interacts specifically
with hsp 27, 60, and 70, angiostatin interacts predominantly with hsp 27 and to some extent with hsp 70; (ii) plasminogen
only partially displaces angiostatin’s binding to hsp 27 and (iii) actin only partially displaces plasminogen/angiostatin
binding to hsps. It is conceivable therefore that surface-associated hsps could mediate the binding of these ligands to cells
like ECs. 相似文献
13.
Jean-Marie Blanchard 《Biochemical and biophysical research communications》1980,97(2):524-529
By using Adenovirus 2 infected HeLa cells labeled during very brief pulses of (3H)Uridine, we have shown that nascent chains of heterogenous nuclear RNA (hnRNA) were already associated with proteins to form ribonucleoprotein particles (hnRNP). It was also shown that the small Ad2 specific VA RNA was not associated with these hnRNP. 相似文献
14.
We used the yeast two-hybrid system to show that the serum response factor (SRF) and zinc-fingers and homeobox 1 (ZHXI) proteins interact with the A subunit of nuclear factor-Y (NF-YA). GST pulldown assays revealed that both proteins interact specifically with NF-YA in vitro. Amino acids located between 272 and 564, a region that contains two homeodomains, are required for the interaction of ZHX1 with NF-YA. Two different domains of NF-YA, a glutamine-rich region and a serine/threonine-rich region, are necessary for the interactions with ZHX1 and SRF, respectively. 相似文献
15.
Envelope membrane proteins that interact with chloroplastic precursor proteins. 总被引:23,自引:5,他引:18
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The post-translational transport of cytoplasmically synthesized precursor proteins into chloroplasts requires proteins in the envelope membranes. To identify some of these proteins, label transfer cross-linking was performed using precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase (prSSU) that was blocked at an early stage of the transport process. Two envelope proteins were identified: an 86-kD protein and a 75-kD protein, both present in the outer membrane. Labeling of both proteins required prSSU and could not be accomplished with SSU lacking a transit peptide. Labeling of the 75-kD protein occurred only when low levels of ATP were present, whereas labeling of the 86-kD protein occurred in the absence of exogenous ATP. Although both labeled proteins were identified as proteins of the outer envelope membrane, the labeled form of the 75-kD protein could only be detected in fractions containing mixed envelope membranes. Based on these observations, we propose that prSSU first binds in an ATP-independent fashion to the 86-kD protein. The energy-requiring step is association with the 75-kD protein and assembly of a translocation contact site between the inner and outer membrane of the chloroplastic envelope. 相似文献
16.
17.
Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis 总被引:14,自引:1,他引:13
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Morita E Sandrin V Chung HY Morham SG Gygi SP Rodesch CK Sundquist WI 《The EMBO journal》2007,26(19):4215-4227
TSG101 and ALIX both function in HIV budding and in vesicle formation at the multivesicular body (MVB), where they interact with other Endosomal Sorting Complex Required for Transport (ESCRT) pathway factors required for release of viruses and vesicles. Proteomic analyses revealed that ALIX and TSG101/ESCRT-I also bind a series of proteins involved in cytokinesis, including CEP55, CD2AP, ROCK1, and IQGAP1. ALIX and TSG101 concentrate at centrosomes and are then recruited to the midbodies of dividing cells through direct interactions between the central CEP55 'hinge' region and GPP-based motifs within TSG101 and ALIX. ESCRT-III and VPS4 proteins are also recruited, indicating that much of the ESCRT pathway localizes to the midbody. Depletion of ALIX and TSG101/ESCRT-I inhibits the abscission step of HeLa cell cytokinesis, as does VPS4 overexpression, confirming a requirement for these proteins in cell division. Furthermore, ALIX point mutants that block CEP55 and CHMP4/ESCRT-III binding also inhibit abscission, indicating that both interactions are essential. These experiments suggest that the ESCRT pathway may be recruited to facilitate analogous membrane fission events during HIV budding, MVB vesicle formation, and the abscission stage of cytokinesis. 相似文献
18.
19.
The fluorescent probe 1-anilinonaphthalene 8-sulfonate was used to examine the binding of spin-labeled local anesthetics to lipid model systems, to the membranes of human red blood cells, and rabbit sarcoplasmic reticulum. 1-Anilinonaphthalene 8-sulfonate exhibits two distinct fluorescent lifetimes when bound to these biological membranes. The shorter lifetime represents the probe associated with the purely lipid region while the longer lifetime is associated with the protein region. The spin-labeled local anesthetic quenches the fluorescence of both of these components as indicated by the decrease in the lifetimes. Since nitroxide free radicals are known to quench fluorophores upon 'contract', the results reflect the relative interaction of local anesthetics with membrane lipids and proteins. The evidence is consistent with the concept of multiple binding sites for local anesthetics in membranes. Local anesthetics, once intercalated into the bilayer, may diffuse laterally and interact with membrane components, lipid as well as proteins. In biological membranes, however, positively charged local anesthetics are better able to quench 1-anilinonaphthalene 8-sulfonate in protein regions, suggesting that the interaction between local anesthetics and membrane proteins can be electrostatic in nature. 相似文献