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1.
Maowulan Maimaitiyiming Haruko Kumanogoh Shun Nakamura Mitsuhiro Morita Shohei Maekawa 《Protein expression and purification》2013,87(2):67-71
Septin forms a conserved family of cytoskeletal GTP-binding proteins that have diverse roles in protein scaffolding, vesicle trafficking and cytokinesis. There are 14 mammalian septin isoforms and these isoforms assemble into hetero-oligomeric rod-shaped complexes and these short filaments are the basal units to construct higher-order structures such as longer filaments, rings, gauzes or hourglasses. Septin expressed in a eukaryotic expression system forms various structures such as bundles, sheets, helixes, and rings. Septin expressed in bacteria formed hexameric short filaments and single or parallel long filaments, but no such higher order structures were observed so far. In a previous study, we showed maturation-dependent localization of septin isoforms to the lipid raft fraction of rat brain. In this study, we attempted further purification of raft-localized septin isoforms. Repeated cycles of extraction with high MgCl2 solution and precipitation under low ionic solution were combined with several column procedures. The obtained fraction contained several septin isoforms and showed rings of bundled filaments with a diameter of ~0.4 μm. Several non-septin proteins were also detected in the fraction. We also attempted expression of septin isoforms in bacteria and found that the expressed septin complexes formed bundles of filaments. In addition to linear and curled filaments, circular bundles of thin filaments with a diameter of ~0.6 μm were also observed. These results suggest that the curvature of the bundles of septin filaments may be regulated by the regulatory factor(s) in the lipid raft. 相似文献
2.
Septin filaments form ordered hourglass and ring-shaped structures in close apposition to the yeast bud-neck membrane. The septin hourglass scaffolds the asymmetric localization of many essential cell division proteins. However, it is unknown whether the septin structures have an overall polarity along the mother-daughter axis that determines the asymmetric protein localization. Here we engineered rigid septin- green fluorescent protein (GFP) fusions with various fluorescence dipole directions by changing the position of the GFP beta-barrel relative to the septin filament axis. We then used polarized fluorescence microscopy to detect potential asymmetries in the filament organization. We found that both the hourglass and ring filament assemblies have sub-resolution C(2) symmetry and lack net polarity along the mother-daughter axis. The hourglass filaments have an additional degree of symmetry relative to the ring filaments, most likely due to a twist in their higher-order structure. We previously reported that during the hourglass to rings transition septin filaments change their direction. Here we show that the filaments also undergo a change in their lateral organization, consistent with filament untwisting. The lack of net septin polarity along the mother-daughter axis suggests that there are no septin-based structural reasons for the observed asymmetry of other proteins. We discuss possible anisotropic processes that could break the septin symmetry and establish the essential bud-neck asymmetry. 相似文献
3.
mRNA trafficking in fungi 总被引:1,自引:0,他引:1
4.
Epithelial polarity requires septin coupling of vesicle transport to polyglutamylated microtubules
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In epithelial cells, polarized growth and maintenance of apical and basolateral plasma membrane domains depend on protein sorting from the trans-Golgi network (TGN) and vesicle delivery to the plasma membrane. Septins are filamentous GTPases required for polarized membrane growth in budding yeast, but whether they function in epithelial polarity is unknown. Here, we show that in epithelial cells septin 2 (SEPT2) fibers colocalize with a subset of microtubule tracks composed of polyglutamylated (polyGlu) tubulin, and that vesicles containing apical or basolateral proteins exit the TGN along these SEPT2/polyGlu microtubule tracks. Tubulin-associated SEPT2 facilitates vesicle transport by maintaining polyGlu microtubule tracks and impeding tubulin binding of microtubule-associated protein 4 (MAP4). Significantly, this regulatory step is required for polarized, columnar-shaped epithelia biogenesis; upon SEPT2 depletion, cells become short and fibroblast-shaped due to intracellular accumulation of apical and basolateral membrane proteins, and loss of vertically oriented polyGlu microtubules. We suggest that septin coupling of the microtubule cytoskeleton to post-Golgi vesicle transport is required for the morphogenesis of polarized epithelia. 相似文献
5.
Senthil-Kumar Devan Stephan Schott-Verdugo Kira Müntjes Lilli Bismar Jens Reiners Eymen Hachani Lutz Schmitt Astrid Hppner Sander HJ Smits Holger Gohlke Michael Feldbrügge 《PLoS genetics》2022,18(6)
Spatiotemporal expression can be achieved by transport and translation of mRNAs at defined subcellular sites. An emerging mechanism mediating mRNA trafficking is microtubule-dependent co-transport on shuttling endosomes. Although progress has been made in identifying various components of the endosomal mRNA transport machinery, a mechanistic understanding of how these RNA-binding proteins are connected to endosomes is still lacking. Here, we demonstrate that a flexible MademoiseLLE (MLLE) domain platform within RNA-binding protein Rrm4 of Ustilago maydis is crucial for endosomal attachment. Our structure/function analysis uncovered three MLLE domains at the C-terminus of Rrm4 with a functionally defined hierarchy. MLLE3 recognises two PAM2-like sequences of the adaptor protein Upa1 and is essential for endosomal shuttling of Rrm4. MLLE1 and MLLE2 are most likely accessory domains exhibiting a variable binding mode for interaction with currently unknown partners. Thus, endosomal attachment of the mRNA transporter is orchestrated by a sophisticated MLLE domain binding platform. 相似文献
6.
Mammalian septins are a family of guanosine triphosphate-binding proteins thought to play a role in a number of key cellular processes, such as cytokinesis, protein scaffolding and vesicle trafficking. Although their precise functions remain to be determined, electron microscopy has shown septin filament formation in vitro and a role as a cytoskeletal polymer has been proposed. Here, we present a 3D reconstruction of septin filaments determined using electron microscopy of negatively stained specimens and single-particle image processing. Septin was isolated from rat brain as an approximately 240-kDa complex, from which immunoblotting and N-terminal sequencing identified the major components as septins 3, 5 and 7. Electron microscopy and single-particle analysis indicated that the majority of the septin filaments were ∼ 27 nm long. A comparison of 3D volumes obtained using two independent starting models (a row of spheres or a helix) and projection matching techniques revealed no major differences at the final resolution of 27 Å, and this structure was highly reproducible when the entire procedure was repeated several times. The reconstruction revealed three apparent subunits, each separated by a cleft; these subunits were similar, but not identical, possibly indicating multiple isoforms within each filament. In some views a smaller cleft appeared to separate the subunits into two smaller regions, perhaps reflecting the presence of septin dimers. This is the first 3D reconstruction of the native septin assembly, and appears compatible with the hypothesis that the septin complex is a hexamer consisting of dimers or heterotrimers. Further investigations are necessary to confirm how the structure of the filaments determined in the present study correlates with the roles of septins in vivo. 相似文献
7.
Interest in the biology of mammalian septin proteins has undergone a birth in recent years. Originally identified as critical for yeast budding throughout the 1970s, the septin family is now recognized to extend from yeast to humans and is associated with a variety of events ranging from cytokinesis to vesicle trafficking. An emerging theme for septins is their presence at sites where active membrane or cytoplasmic partitioning is occurring. Here, we briefly review the mammalian septin protein family and focus on a prototypic human and mouse septin, termed SEPT5, that is expressed in the brain, heart, and megakaryocytes. Work from neurobiology laboratories has linked SEPT5 to the exocytic complex of neurons, with implications that SEPT5 regulates neurotransmitter release. Striking similarities exist between neurotransmitter release and the platelet-release reaction, which is a critical step in platelet response to vascular injury. Work from our laboratory has characterized the platelet phenotype from mice containing a targeted deletion of SEPT5. Most strikingly, platelets from SEPT5(null) animals aggregate and release granular contents in response to subthreshold levels of agonists. Thus, the characterization of a SEPT5-deficient mouse has linked SEPT5 to the platelet exocytic process and, as such, illustrates it as an important protein for regulating platelet function. Recent data suggest that platelets contain a wide repertoire of different septin proteins and assemble to form macromolecular septin complexes. The mouse platelet provides an experimental framework to define septin function in hemostasis, with implications for neurobiology and beyond. 相似文献
8.
Radka Káčeriková Jana Godočíková Zhexin Wang Eva Kutejová Stefan Raunser Marian Farkašovský 《Biologia》2018,73(10):1025-1033
Septins are a family of eukaryotic guanosine phosphate-binding proteins that form linear heterooligomeric complexes, which, in turn, polymerize end-on-end into filaments. These filaments further assemble into higher-order structures at distinct subcellular locations. Dynamic changes in the organization of septin cortex structures appear during cell cycle progression. A variety of regulatory proteins and posttranslational modifications are involved in changes to the structure of septin assemblies during the entire cell cycle. In particular, septin-associated protein kinases mediate changes to septin higher order structures or interconnect cellular morphogenesis with the cell cycle. Yeast cyclin-dependent kinase, a master cell cycle regulator, is required for the initiation of a new septin ring. Here, using epifluoresence and electron microscopy, we show that upon phosphorylation by the Cdc28 kinase, septin filaments disassemble into hetero-octameric building blocks, and that filament depolymerization is specifically G1 cyclin-dependent. 相似文献
9.
In eukaryotic cells, proteins are targeted to their final subcellular locations with precise timing. A key underlying mechanism is the active transport of cognate mRNAs, which in many systems can be linked intimately to membrane trafficking. A prominent example is the long‐distance endosomal transport of mRNAs and their local translation. Here, we describe current highlights of fundamental mechanisms of the underlying transport process as well as of biological functions ranging from endosperm development in plants to fungal pathogenicity and neuronal processes. Translation of endosome‐associated mRNAs often occurs at the cytoplasmic surface of endosomes, a process that is needed for membrane‐assisted formation of heteromeric protein complexes and for accurate subcellular targeting of proteins. Importantly, endosome‐coupled translation of mRNAs encoding mitochondrial proteins, for example, seems to be particularly important for efficient organelle import and for regulating subcellular mitochondrial activity. In essence, these findings reveal a new mechanism of loading newly synthesised proteins onto endocytic membranes enabling intimate crosstalk between organelles. The novel link between endosomes and mitochondria adds an inspiring new level of complexity to trafficking and organelle biology. 相似文献
10.
Nagata K Kawajiri A Matsui S Takagishi M Shiromizu T Saitoh N Izawa I Kiyono T Itoh TJ Hotani H Inagaki M 《The Journal of biological chemistry》2003,278(20):18538-18543
Septins are a family of conserved proteins implicated in a variety of cellular functions such as cytokinesis and vesicle trafficking, but their properties and modes of action are largely unknown. Here we now report findings of immunocytochemical and biochemical characterization of a mammalian septin, MSF-A. Using an antibody specific for MSF subfamily proteins, MSF-A was found to be expressed predominantly in mammary human mammary epithelial cells (HMEC). MSF-A was associated with microtubules in interphase HMEC cells as it localized with the mitotic spindle and the bundle of microtubule at midzone during mitosis. Biochemical analysis revealed direct binding of MSF-A with polymerized tubulin through its central region containing guanine nucleotide-interactive motifs. GTPase activity, however, was not required for the association. Conditions that disrupt the microtubule network also disrupted the MSF-A-containing filament structure, resulting in a punctate cytoplasmic pattern. Depletion of MSF-A using small interfering RNAs caused incomplete cell division and resulted in the accumulation of binucleated cells. Unlike Nedd5, an MSF mutant deficient in GTPase activity forms filament indistinguishable from that of the wild type in COS cells. These results strongly suggest that septin filaments may interact not only with actin filaments but also with microtubule networks and that GTPase activity of MSF-A is not indispensable to incorporation of MSF-A into septin filaments. 相似文献
11.
《Fungal Biology Reviews》2020,34(2):89-99
Membrane-coupled RNA transport is an emerging theme in fungal biology. This review focuses on the RNA cargo and mechanistic details of transport via two inter-related sets of organelles: endosomes and extracellular vesicles for intra- and intercellular RNA transfer. Simultaneous transport and translation of messenger RNAs (mRNAs) on the surface of shuttling endosomes is a conserved process pertinent to highly polarised eukaryotic cells, such as hyphae or neurons. Here we detail the endosomal mRNA transport machinery components and mRNA targets of the core RNA-binding protein Rrm4. Extracellular vesicles (EVs) are newly garnering interest as mediators of intercellular communication, especially between pathogenic fungi and their hosts. Landmark studies in plant–fungus interactions indicate EVs as a means of delivering various cargos, most notably small RNAs (sRNAs), for cross-kingdom RNA interference. Recent advances and implications of the nascent field of fungal EVs are discussed and potential links between endosomal and EV-mediated RNA transport are proposed. 相似文献
12.
septin基因家族的研究进展 总被引:1,自引:0,他引:1
septin是一个广泛存在于除植物以外所有真核生物中的基因家族。最初认为septin家族是与酵母细胞胞质分裂相关的基因家族。然而随着研究的深入, 人们发现这类基因编码的蛋白质在许多生物体内出现了较大的功能分化, 尤其在哺乳动物细胞中, 他们不仅成员众多, 且参与了细胞分裂、细胞极化、囊泡运输及胞膜重构等多个过程。更引起研究人员重视的是: 最近有大量数据表明, 这一家族的一些成员与肿瘤发生、神经功能障碍和病原微生物感染的过程直接相关。因此, 近年来septin家族的功能研究正逐步成为细胞生物学及病理学研究的新热点。文章将试图从septin基因家族的种类、结构特点、生物学功能及其与人类疾病的关系等方面的研究进展进行综述。 相似文献
13.
Borg/septin interactions and the assembly of mammalian septin heterodimers,trimers, and filaments 总被引:9,自引:0,他引:9
Sheffield PJ Oliver CJ Kremer BE Sheng S Shao Z Macara IG 《The Journal of biological chemistry》2003,278(5):3483-3488
Septins constitute a family of guanine nucleotide-binding proteins that were first discovered in the yeast Saccharomyces cerevisiae but are also present in many other eukaryotes. In yeast they congregate at the bud neck and are required for cell division. Their function in metazoan cells is uncertain, but they have been implicated in exocytosis and cytokinesis. Septins have been purified from cells as hetero-oligomeric filaments, but their mechanism of assembly is unknown. Further studies have been limited by the difficulty in expressing functional septin proteins in bacteria. We now show that stable, soluble septin heterodimers can be produced by co-expression from bicistronic vectors in bacteria and that the co-expression of three septins results in their assembly into filaments. Pre-assembled dimers and trimers bind guanine nucleotide and show a slow GTPase activity. The assembly of a heterodimer from monomers in vitro is accompanied by GTP hydrolysis. Borg3, a downstream effector of the Cdc42 GTPase, binds specifically to a septin heterodimer composed of Sept6 and Sept7 and to the Sept2/6/7 trimer, but not to septin monomers or to other heterodimers. Septins associate through their C-terminal coiled-coil domains, and Borg3 appears to recognize the interface between these domains in Sept6 and Sept7. 相似文献
14.
Mostowy S Bonazzi M Hamon MA Tham TN Mallet A Lelek M Gouin E Demangel C Brosch R Zimmer C Sartori A Kinoshita M Lecuit M Cossart P 《Cell host & microbe》2010,8(5):433-444
Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens. 相似文献
15.
16.
Pissuti Damalio JC Garcia W Alves Macêdo JN de Almeida Marques I Andreu JM Giraldo R Garratt RC Ulian Araújo AP 《Biochimie》2012,94(3):628-636
Septins are a conserved group of GTP-binding proteins that form hetero-oligomeric complexes which assemble into filaments. These are essential for septin function, including their role in cytokinesis, cell division, exocytosis and membrane trafficking. Septin 2 (SEPT2) is a member of the septin family and has been associated with neurofibrillary tangles and other pathological features of senile plaques in Alzheimer's disease. An in silico analysis of the amino acid sequence of SEPT2 identified regions with a significant tendency to aggregate and/or form amyloid. These were all observed within the GTP-binding domain. This was consistent with the experimental identification of a structure rich in β-sheet during temperature induced unfolding transitions observed for both the full length protein and the GTP-binding domain alone. This intermediate state is characterized by irreversible aggregation and has the ability to bind Thioflavin-T, suggesting its amyloid nature. Under electron microscopy, fibers extending for several micrometers in length could be visualized. The results shown in this study support the hypothesis that single septins, when present in excess or with unbalanced stoichiometries, may be unstable and assemble into amyloid-like structures. 相似文献
17.
Septins are a conserved family of eukaryotic GTP-binding, filament-forming proteins. In Saccharomyces cerevisiae, five septins (Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Shs1p) form a complex and colocalize to the incipient bud site and as a collar of filaments at the neck of budded cells. Septins serve as a scaffold to localize septin-associated proteins involved in diverse processes and as a barrier to diffusion of membrane-associated proteins. Little is known about the role of nucleotide binding in septin function. Here, we show that Cdc3p, Cdc10p, Cdc11p, and Cdc12p all bind GTP and that P-loop and G4 motif mutations affect nucleotide binding and result in temperature-sensitive defects in septin localization and function. Two-hybrid, in vitro, and in vivo analyses show that for all four septins nucleotide binding is important in septin-septin interactions and complex formation. In the absence of complete complexes, septins do not localize to the cortex, suggesting septin localization factors interact only with complete complexes. When both complete and partial complexes are present, septins localize to the cortex but do not form a collar, perhaps because of an inability to form filaments. We find no evidence that nucleotide binding is specifically involved in the interaction of septins with septin-associated proteins. 相似文献
18.
The septins are a family of GTPases involved in cytokinesis in budding yeast, Drosophila, and vertebrates (see for review). Septins are associated with a system of 10 nm filaments at the S. cerevisiae bud neck, and heteromultimeric septin complexes have been isolated from cell extracts in a filamentous state. A number of septins have been shown to bind and hydrolyze guanine nucleotide. However, the role of GTP binding and hydrolysis in filament formation has not been elucidated. Furthermore, several lines of evidence suggest that not all the subunits of the septin complex are required for all aspects of septin function. To address these questions, we have reconstituted filament assembly in vitro by using a recombinant Xenopus septin, Xl Sept2. Filament assembly is GTP dependent; moreover, the coiled-coil domain common to most septins is not essential for filament formation. Septin polymerization is preceded by a lag phase, suggesting a cooperative assembly mechanism. The slowly hydrolyzable GTP analog, GTP-gamma-S, also induces polymerization, indicating that polymerization does not require GTP hydrolysis. If the properties of Xl Sept2 filaments reflect those of native septin complexes, these results imply that the growth or stability of septin filaments, or both, is regulated by the state of bound nucleotide. 相似文献
19.
Bertin A McMurray MA Thai L Garcia G Votin V Grob P Allyn T Thorner J Nogales E 《Journal of molecular biology》2010,404(4):711-731
Septins are a conserved family of GTP-binding proteins that assemble into symmetric linear heterooligomeric complexes, which in turn are able to polymerize into apolar filaments and higher-order structures. In budding yeast (Saccharomyces cerevisiae) and other eukaryotes, proper septin organization is essential for processes that involve membrane remodeling, such as the execution of cytokinesis. In yeast, four septin subunits form a Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 heterooctameric rod that polymerizes into filaments thought to form a collar around the bud neck in close contact with the inner surface of the plasma membrane. To explore septin-membrane interactions, we examined the effect of lipid monolayers on septin organization at the ultrastructural level using electron microscopy. Using this methodology, we have acquired new insights into the potential effect of septin-membrane interactions on filament assembly and, more specifically, on the role of phosphoinositides. Our studies demonstrate that budding yeast septins interact specifically with phosphatidylinositol-4,5-bisphosphate (PIP2) and indicate that the N terminus of Cdc10 makes a major contribution to the interaction of septin filaments with PIP2. Furthermore, we found that the presence of PIP2 promotes filament polymerization and organization on monolayers, even under conditions that prevent filament formation in solution or for mutants that prevent filament formation in solution. In the extreme case of septin complexes lacking the normally terminal subunit Cdc11 or the normally central Cdc10 doublet, the combination of the PIP2-containing monolayer and nucleotide permitted filament formation in vitro via atypical Cdc12-Cdc12 and Cdc3-Cdc3 interactions, respectively. 相似文献
20.
《The Journal of cell biology》1996,133(3):605-616
Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells. 相似文献