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At least 19 recordsLinked to original sources

PubMed2004

Human septin-septin interactions as a prerequisite for targeting septin complexes in the cytosol.

Septins are a cytosolic GTP-binding protein family first characterized in yeast, but gaining increasing recognition as critical protagonists in higher eukaryotic cellular events. Mammalian septins have been associated with cytokinesis and exocytosis, along with contributing to the development of neurological disorders. Ten different septins, divided into four groups, have been identified in mammals, and individual septins are capable of interacting with each other to form macromolecular complexes. The present study characterizes the structural requirements for human septin-septin interactions using a yeast two-hybrid system. We focus on three septins that are highly expressed in platelets and neurons, SEPT4 [previously designated H5, CDCrel-2 (cell-division-control-related-2), PNUTL2], SEPT5 (CDCrel-1, PNUTL1) and SEPT8 (KIAA0202). Each of these three septins contains a characteristic domain structure consisting of unique N- and C-termini, and a central core domain conserved among the family of proteins. The yeast two-hybrid system yielded data consistent with a model where each of the three septins can interact with itself (homotypic assembly) or with one of the other septins (heterotypic assembly). For SEPT5 and SEPT8, the results illustrate a model whereby heterotypic septin assembly is dependent on the conserved central core domain and homotypic interactions require the N- and C-termini of each protein. We also characterized a model in which the proper cellular localization of SEPT5 and SEPT8 requires concomitant expression of both proteins. Co-transfection of SEPT5 and SEPT8 results in both proteins targeted to a vesicular-like location. Therefore the cellular repertoire of human septins has an impact on function by targeting septin macromolecular complexes to specific cellular locations.

Amino Acid Sequence↗

PubMed1998

Role of the yeast Gin4p protein kinase in septin assembly and the relationship between septin assembly and septin function.

To identify septin-interacting proteins in Saccharomyces cerevisiae, we screened for mutations that are synthetically lethal with a cdc12 septin mutation. One of the genes identified was GIN4, which encodes a protein kinase related to Hsl1p/Nik1p and Ycl024Wp in S. cerevisiae and to Nim1p/Cdr1p and Cdr2p in Schizosaccharomyces pombe. The Gin4p kinase domain displayed a two-hybrid interaction with the COOH-terminal portion of the Cdc3p septin, and Gin4p colocalized with the septins at the mother-bud neck. This localization depended on the septins and on the COOH-terminal (nonkinase) region of Gin4p, and overproduction of this COOH-terminal region led to a loss of septin organization and associated morphogenetic defects. We detected no effect of deleting YCL024W, either alone or in combination with deletion of GIN4. Deletion of GIN4 was not lethal but led to a striking reorganization of the septins accompanied by morphogenetic abnormalities and a defect in cell separation; however, remarkably, cytokinesis appeared to occur efficiently. Two other proteins that localize to the neck in a septin-dependent manner showed similar reorganizations and also appeared to remain largely functional. The septin organization observed in gin4Delta vegetative cells resembles that seen normally in cells responding to mating pheromone, and no Gin4p was detected in association with the septins in such cells. The organization of the septins observed in gin4Delta cells and in cells responding to pheromone appears to support some aspects of the model for septin organization suggested previously by Field et al. (Field, C.M., O. Al-Awar, J. Rosenblatt, M.L. Wong, B. Alberts, and T.J. Mitchison. 1996. J. Cell Biol. 133:605-616).

Amino Acid Sequence↗

PubMed2000

Phosphorylation of a new brain-specific septin, G-septin, by cGMP-dependent protein kinase.

The septins are a family of GTPase enzymes, some of which are required for the cytokinesis stage of cell division and others of which are associated with exocytosis. We purified and cloned the cDNA for a 40-kDa protein from rat brain that is a substrate for type I cGMP-dependent protein kinase (PKG). The amino acid sequences of two tryptic peptides of P40 showed high homology to the septins. Molecular cloning revealed the 358-amino acid P40 to be a new member of the septin family. P40 was named G-septin, as it is phosphorylated in vitro by PKG, but relatively poorly by the related cAMP-dependent protein kinase and not by protein kinase C. Two splice variants of G-septin (alpha and beta) were found with distinct N and C termini, but a common GTPase domain. G-septin lacks the C-terminal coiled-coil domain characteristic of all other mammalian septins and uniquely has two predicted phosphorylation site motifs for type I PKG. Photoaffinity labeling with [alpha-(32)P]GTP confirmed that G-septin is a GTP-binding protein. Northern blotting showed that G-septin mRNA (5.0 kilobases) is highly expressed in brain and undetectable in 12 other tissues, indicating that the G-septins are primarily neuronal proteins. Very low levels of 6.0-, 3.4-, and 2.6-kilobase transcripts were found in testis. Our results reveal a new class of brain-specific septins that may be regulated by PKG in neurons.

Alternative Splicing↗

PubMed2003

Cloning and functional characterization of human septin 10, a novel member of septin family cloned from dendritic cells.

Septins are a family of cytoskeletal proteins with GTPase activity involved in various cellular biological processes. Here, we describe the identification of septin 10, a novel septin family member from human dendritic cells. The 3018-bp full-length cDNA potentially encodes a 517-residue peptide, sharing closest homology to human septin 8 and septin 6. With a conserved GTP-binding motif at its N-terminus, septin 10 protein can bind to GTP and exert GTPase activity. Human septin 10 gene is electronically mapped to 8q11.2-12. It is ubiquitously expressed in normal tissues, with the abundant expression in heart and kidney, placenta, skeletal muscles, liver and lung, as well as various tumor cell lines. Interestingly, dendritic cells express upregulated septin 10 upon LPS-induced maturation. Based on its GFP-fusion protein, septin 10 is found to localize in cytoplasm and nucleus, with a subcellular pattern independent of the filamentous state of actin.

Amino Acid Sequence↗

PubMed2004

Protein-protein interactions governing septin heteropentamer assembly and septin filament organization in Saccharomyces cerevisiae.

Mitotic yeast (Saccharomyces cerevisiae) cells express five related septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) that form a cortical filamentous collar at the mother-bud neck necessary for normal morphogenesis and cytokinesis. All five possess an N-terminal GTPase domain and, except for Cdc10, a C-terminal extension (CTE) containing a predicted coiled coil. Here, we show that the CTEs of Cdc3 and Cdc12 are essential for their association and for the function of both septins in vivo. Cdc10 interacts with a Cdc3-Cdc12 complex independently of the CTE of either protein. In contrast to Cdc3 and Cdc12, the Cdc11 CTE, which recruits the nonessential septin Shs1, is dispensable for its function in vivo. In addition, Cdc11 forms a stoichiometric complex with Cdc12, independent of its CTE. Reconstitution of various multiseptin complexes and electron microscopic analysis reveal that Cdc3, Cdc11, and Cdc12 are all necessary and sufficient for septin filament formation, and presence of Cdc10 causes filament pairing. These data provide novel insights about the connectivity among the five individual septins in functional septin heteropentamers and the organization of septin filaments.

Amino Acid Sequence↗

PubMed2002

Bni5p, a septin-interacting protein, is required for normal septin function and cytokinesis in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7Delta strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7Delta mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.

Carrier Proteins↗

PubMed1998

Polymerization of purified yeast septins: evidence that organized filament arrays may not be required for septin function.

The septins are a family of proteins required for cytokinesis in a number of eukaryotic cell types. In budding yeast, these proteins are thought to be the structural components of a filament system present at the mother-bud neck, called the neck filaments. In this study, we report the isolation of a protein complex containing the yeast septins Cdc3p, Cdc10p, Cdc11p, and Cdc12p that is capable of forming long filaments in vitro. To investigate the relationship between these filaments and the neck filaments, we purified septin complexes from cells deleted for CDC10 or CDC11. These complexes were not capable of the polymerization exhibited by wild-type preparations, and analysis of the neck region by electron microscopy revealed that the cdc10Delta and cdc11Delta cells did not contain detectable neck filaments. These results strengthen the hypothesis that the septins are the major structural components of the neck filaments. Surprisingly, we found that septin dependent processes like cytokinesis and the localization of Bud4p to the neck still occurred in cdc10Delta cells. This suggests that the septins may be able to function in the absence of normal polymerization and the formation of a higher order filament structure.

Cell Cycle Proteins↗

PubMed2003

Molecular dissection of a yeast septin: distinct domains are required for septin interaction, localization, and function.

The septins are a family of cytoskeletal proteins present in animal and fungal cells. They were first identified for their essential role in cytokinesis, but more recently, they have been found to play an important role in many cellular processes, including bud site selection, chitin deposition, cell compartmentalization, and exocytosis. Septin proteins self-associate into filamentous structures that, in yeast cells, form a cortical ring at the mother bud neck. Members of the septin family share common structural domains: a GTPase domain in the central region of the protein, a stretch of basic residues at the amino terminus, and a predicted coiled-coil domain at the carboxy terminus. We have studied the role of each domain in the Saccharomyces cerevisiae septin Cdc11 and found that the three domains are responsible for distinct and sometimes overlapping functions. All three domains are important for proper localization and function in cytokinesis and morphogenesis. The basic region was found to bind the phosphoinositides phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate. The coiled-coil domain is important for interaction with Cdc3 and Bem4. The GTPase domain is involved in Cdc11-septin interaction and targeting to the mother bud neck. Surprisingly, GTP binding appears to be dispensable for Cdc11 function, localization, and lipid binding. Thus, we find that septins are multifunctional proteins with specific domains involved in distinct molecular interactions required for assembly, localization, and function within the cell.

Amino Acid Sequence↗

PubMed1999

Characterization of the mammalian septin H5: distinct patterns of cytoskeletal and membrane association from other septin proteins.

The mechanisms controlling cytokinesis during yeast budding and animal cell fission appear quite different, yet both require members of the septin protein family. Mammalian homologs of this novel family of GTPases have been identified but little is known about their properties or functions. Using an antibody specific for the mammalian septin H5, we show that this protein is expressed at distinct levels in a variety of tissues. Tissue expression levels in different tissues did not coincide with those of the only previously characterized mammalian septin Nedd5. H5, like Nedd5, localizes to the cleavage furrow in mitotic fibroblast cells but in non-mitotic cells these proteins associate with actin filaments in different ways. Nedd5 predominantly localizes with stress fibers, but only associates with central portions of the microfilament bundles. In contrast, H5 associates with the entire length of the stress fibers and the cortical actin network. Conditions that disrupt the actin cytoskeleton also disrupt the filamentous patterns of both Nedd5 and H5, resulting in a punctate cytoplasmic pattern. Cell fractionation revealed that H5 co-fractionated with actin, while Nedd5 was predominantly restricted to the membrane fraction. Co-immunoprecipitation experiments revealed that although H5 will co-precipitate with Nedd5, the precipitation is not quantitative. Taken together, these results not only show that H5 behaves like a septin, but also demonstrate that individual septin proteins have distinct properties, suggesting that they may play different roles in cytokinesis and in other stages of the cell cycle.

3T3 Cells↗

PubMed1996

Identification of a developmentally regulated septin and involvement of the septins in spore formation in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae CDC3, CDC10, CDC11, and CDC12 genes encode a family of related proteins, the septins, which are involved in cell division and the organization of the cell surface during vegetative growth. A search for additional S. cerevisiae septin genes using the polymerase chain reaction identified SPR3, a gene that had been identified previously on the basis of its sporulation-specific expression. The predicted SPR3 product shows 25-40% identity in amino acid sequence to the previously known septins from S. cerevisiae and other organisms. Immunoblots confirmed the sporulation-specific expression of Spr3p and showed that other septins are also present at substantial levels in sporulating cells. Consistent with the expression data, deletion of SPR3 in either of two genetic backgrounds had no detectable effect on exponentially growing cells. In one genetic background, deletion of SPR3 produced a threefold reduction in sporulation efficiency, although meiosis appeared to be completed normally. In this background, deletion of CDC10 had no detectable effect on sporulation. In the other genetic background tested, the consequences of the two deletions were reversed. Immunofluorescence observations suggest that Spr3p, Cdc3p, and Cdc11p are localized to the leading edges of the membrane sacs that form near the spindle-pole bodies and gradually extend to engulf the nuclear lobes that contain the haploid chromosome sets, thus forming the spores. Deletion of SPR3 does not prevent the localization of Cdc3p and Cdc11p, but these proteins appear to be less well organized, and the intensity of their staining is reduced. Taken together, the results suggest that the septins play important but partially redundant roles during the process of spore formation.

Amino Acid Sequence↗

PubMed2002

Phosphorylation of the septin cdc3 in g1 by the cdc28 kinase is essential for efficient septin ring disassembly.

The septins constitute a family of filament-forming proteins ubiquitous in eukaryotic species. We demonstrate here that the Saccharomyces cerevisiae septin, Cdc3, is a substrate of the cell cycle regulatory cyclin-dependent kinase (Cdk), Cdc28. Two serines near the C-terminus of Cdc3 are phosphorylated in a Cdc28-dependent manner. Analysis of a mutant allele that cannot be phosphorylated at these sites revealed an effect of Cdc28 phosphorylation of Cdc3 at the time of budding. Immunofluorescence analysis of wild-type and mutant Cdc3 indicated that prevention of phosphorylation at Cdc28-dependent sites impairs the disassembly of the old septin ring, which is inherited at mitosis but which usually disappears immediately prior to assembly of a new ring. Furthermore, immuno-fluorescence analysis of septin ring dynamics in a G1 cyclin (Cln) mutant suggests that G1 cyclin function is required for efficient ring disassembly. Thus, phosphorylation of Cdc3 by the Cdc28 kinase at the end of G1 may facilitate initiation of a new cell cycle by promoting disassembly of the obsolete septin ring from the previous cell cycle.

Amino Acid Sequence↗

PubMed2002

Human septin-septin interaction: CDCrel-1 partners with KIAA0202.

Septins are evolutionary conserved cytoskeletal GTPases forming heteropolymer complexes involved in cytokinesis and other cellular processes. CDCrel-1 (cell division cycle related-1) is a recently cloned and characterized human septin which is highly expressed in non-dividing cells, such as neurons. Using a yeast two-hybrid system we demonstrate that CDCrel-1 partners with another uncharacterized human septin, KIAA0202. The interaction of CDCrel-1 and KIAA0202 was confirmed in the human leukemia cell line K-562 using pull-down assays with a KIAA0202-glutathione S-transferase fusion protein and by immunoprecipitation of the CDCrel-1-KIAA0202 complex with an anti-KIAA0202 antibody. Expression studies of the two human septins revealed a concomitant expression of both proteins in certain cells.

Antibodies↗

PubMed2004

Septin 3 (G-septin) is a developmentally regulated phosphoprotein enriched in presynaptic nerve terminals.

The septins are GTPase enzymes with multiple roles in cytokinesis, cell polarity or exocytosis. The proteins from the mammalian septin genes are called Sept1-10. Most are expressed in multiple tissues, but the mRNA for Sept5 (CDCrel-1) and Sept3 (G-septin) appear to be primarily expressed in brain. Sept3 is phosphorylated by cGMP-dependent protein kinase I (PKG-I) and the cGMP/PKG pathway is involved in presynaptic plasticity. Therefore to determine whether Sept3 specifically associates with neurones and nerve terminals we investigated its distribution in rat brain and neuronal cultures. Sept3 protein was detected only in brain by immunoblot, but not in 12 other tissues examined. Levels were high in all adult brain regions, and reduced in those enriched in white matter. Expression was developmentally regulated, being absent in the early embryo, low in late embryonic rat brain and increasing after birth. Like dynamin I, Sept3 was specifically enriched in synaptosomes compared with whole brain, and was only found in a peripheral membrane extract and not in the soluble or membrane extracts. Sept3 was particularly abundant in mossy fibre nerve terminals in the hippocampus. In primary cultured hippocampal neurones Sept3 immunoreactivity was punctate in neurites and predominantly localized to presynaptic terminals, strongly colocalizing with synaptophysin and dynamin I. The specific nerve terminal localization was confirmed by immunogold electron microscopy. Together this shows that Sept3 is a neurone-specific protein highly enriched in nerve terminals which supports a secretory role in synaptic vesicle recycling.

Animals↗

PubMed2003

Ac'septin' a signal: kinase regulation by septins.

Budding yeast monitor shape and the assembly of cytoskeletal structures and convey this information to regulators of cell division, but the molecular mechanisms responsible for monitoring and interpreting spatial information about the cytoskeleton remain poorly understood. A paper in the September issue of Molecular Cell shows that direct binding of components of the septin cytoskeleton may relieve autoinhibition of a conserved checkpoint kinase, creating a simple molecular device for sensing septin cytoskeleton organization.

Animals↗

PubMed2003

The role of Cdc42p GTPase-activating proteins in assembly of the septin ring in yeast.

The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42V36G allele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42V36G or GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth.

Actins↗

PubMed2004

SUMO modification of septin-interacting proteins in Candida albicans.

The initiation of bud and hyphal growth in the opportunistic fungal pathogen Candida albicans both involve polarized morphogenesis. However, there are many differences including the function of the septin proteins, a family of proteins involved in membrane organization in a wide range of organisms. Septins form a characteristic ring on the inner surface of the plasma membrane at the bud neck, whereas the septins are diffusely localized across emerging hyphal tips. In addition, septin rings are maintained at sites of septum formation in hyphae rather than being disassembled immediately after cytokinesis. The possibility that C. albicans septins are regulated by the small ubiquitin-like protein SUMO was examined in this study because the Saccharomyces cerevisiae septins were shown previously to be modified by SUMO (Smt3p). However, SUMO conjugation to septins was not detected during budding or hyphal morphogenesis in C. albicans. These results are supported by the lack of conserved SUMO consensus motifs between septins from the two organisms even after adjusting the predicted Cdc3p and Cdc12p septin sequences to account for mRNA splicing in C. albicans. Interestingly, a homolog of the Smt3p SUMO was identified in the C. albicans genome, and an epitope tagged version of Smt3p was conjugated to a variety of proteins. Immunofluorescence analysis showed prominent Smt3p SUMO localization at bud necks and sites of septum formation in hyphae similar to the septins. However, Smt3p was primarily detected on the mother cell side of the septin ring. A subset of these Smt3p-modified proteins co-immunoprecipitated with the septin Cdc11p. These results indicate that septin-associated proteins and not the septins themselves are the key target of SUMO modification at the bud neck in C. albicans.

Amino Acid Sequence↗

PubMed2005

Interplay between septin organization, cell cycle and cell shape in yeast.

Septins are conserved filament-forming proteins that assemble into cortical cytoskeletal structures in animal and fungal cells. Although rapid progress has been made into the functions of septins, the mechanisms governing their localization and organization remain mysterious. In Saccharomyces cerevisiae, Cdc42p organizes the septin cytoskeleton into a ring in preparation for bud formation, following which septins remain as a collar at the mother-bud neck. We have dissected the phenotype of cdc42(V36T,K94E) cells that display an aberrant cell shape correlated with the development of ectopic septin caps and rings within the bud. The results suggest that a well-assembled septin cortex plays a novel role in directing growth to shape the nascent bud, and that a disorganized septin cortex directs improper growth generating an aberrant neck. Conversely, we found that the elongated bud shape arising as a result of the morphogenesis checkpoint cell cycle delay that accompanies septin perturbation can feed back to exacerbate minor defects in septin organization, by maintaining a bud-tip-localized septin assembly activity that competes with the neck-localized septin cortex. Using this exacerbation as a tool, we uncovered septin organization defects in many mutants not previously known to display such defects, expanding the cast of characters involved in proper assembly of the septin cortex to include CLN1, CLN2, BNI1, BNI4, BUD3, BUD4 and BUD5.

Cell Cycle↗

PubMed2004

Septin ring assembly requires concerted action of polarisome components, a PAK kinase Cla4p, and the actin cytoskeleton in Saccharomyces cerevisiae.

Septins are filament-forming proteins that function in cytokinesis in a wide variety of organisms. In budding yeast, the small GTPase Cdc42p triggers the recruitment of septins to the incipient budding site and the assembly of septins into a ring. We herein report that Bni1p and Cla4p, effectors of Cdc42p, are required for the assembly of the septin ring during the initiation of budding but not for its maintenance after the ring converts to a septin collar. In bni1Delta cla4-75-td mutant, septins were recruited to the incipient budding site. However, the septin ring was not assembled, and septins remained at the polarized growing sites. Bni1p, a formin family protein, is a member of the polarisome complex with Spa2p, Bud6p, and Pea2p. All spa2Delta cla4-75-td, bud6Delta cla4-75-td, and pea2Delta cla4-75-td mutants showed defects in septin ring assembly. Bni1p stimulates actin polymerization for the formation of actin cables. Point mutants of BNI1 that are specifically defective in actin cable formation also exhibited septin ring assembly defects in the absence of Cla4p. Consistently, treatment of cla4Delta mutant with the actin inhibitor latrunculin A inhibited septin ring assembly. Our results suggest that polarisome components and Cla4p are required for the initial assembly of the septin ring and that the actin cytoskeleton is involved in this process.

Actins↗