Characterization of Septin Protein Interactions at the Yeast Bud Neck Using a Tripartite Split GFP Detection System (original) (raw)
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Three-dimensional ultrastructure of the septin filament network inSaccharomyces cerevisiae
Molecular Biology of the Cell, 2011
Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. In budding yeast, five mitotic septins localize at the bud neck, where the plasma membrane is enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2). We previously established the subunit organization within purified yeast septin complexes and how these hetero-octamers polymerize into filaments in solution and on PtdIns4,5P2-containing lipid monolayers. How septin ultrastructure in vitro relates to the septin-containing filaments observed at the neck in fixed cells by thin-section electron microscopy was unclear. A morphological description of these filaments in the crowded space of the cell is challenging, given their small cross section. To examine septin organization in situ, sections of dividing yeast cells were analyzed by electron tomography of freeze-substituted cells, as well as by cryo–electron tomography. We found networks of filaments both perpendicular and parall...
Molecular Biology of The Cell, 2004
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.
Septin Filament Formation Is Essential in Budding Yeast
Developmental Cell, 2011
Septins are GTP-binding proteins that form ordered, rod-like multimeric complexes and polymerize into filaments, but how such supramolecular structure is related to septin function was unclear. In Saccharomyces cerevisiae, four septins form an apolar hetero-octamer (Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11) that associates end-to-end to form filaments. We show that septin filament assembly displays previously unanticipated plasticity. Cells lacking Cdc10 or Cdc11 are able to divide because the now-exposed subunits (Cdc3 or Cdc12, respectively) retain an ability to homodimerize via their so-called G interface, thereby allowing for filament assembly. In such cdc10Δ and cdc11Δ cells, the remaining septins, like wild-type complexes, localize to the cortex at the bud neck and compartmentalize non-septin factors, consistent with a diffusion barrier composed of continuous filaments in intimate contact with the plasma membrane. Conversely, Cdc10 or Cdc11 mutants that cannot self-associate, but "cap" Cdc3 or Cdc12, respectively, prevent filament formation, block cortical localization, and kill cells.
Molecular and Cellular Biology, 2003
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.
Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals
Journal of Cell Biology, 2011
The septins are conserved, GTP-binding proteins important for cytokinesis, membrane compartmentalization, and exocytosis. However, it is unknown how septins are arranged within higher-order structures in cells. To determine the organization of septins in live cells, we developed a polarized fluorescence microscopy system to monitor the orientation of GFP dipole moments with high spatial and temporal resolution. When GFP was fused to septins, the arrangement of GFP dipoles reflected the underlying septin organization. We demonstrated in a filamentous fungus, a budding yeast, and a mammalian epithelial cell line that septin proteins were organized in an identical highly ordered fashion. Fluorescence anisotropy measurements indicated that septin filaments organized into pairs within live cells, just as has been observed in vitro. Additional support for the formation of pairs came from the observation of paired filaments at the cortex of cells using electron microscopy. Furthermore, we ...
Septin structure and function in yeast and beyond
Trends in Cell Biology, 2011
Septins are conserved GTP-binding proteins that assemble into hetero-oligomeric complexes and higher-order structures such as filaments, rings, hourglasses or gauzes. Septins are usually associated with a discrete region of the plasma membrane and function as a cellular scaffold or diffusion barrier to effect cytokinesis, cell polarity, and many other cellular functions. Recent structural studies of septin complexes have provided mechanistic insights into septin filament assembly, but key questions about the assembly, dynamics, and function of different septin cellular structures remain largely unanswered.
Cell cycle control of septin ring dynamics in the budding yeast
Microbiology (Reading, England), 2001
Septins constitute a cytoskeletal structure that is conserved in eukaryotes. In Saccharomyces cerevisiae, the Cdc3, Cdc10, Cdc11, Cdc12 and Shs1/Sep7 septins assemble as a ring that marks the cytokinetic plane throughout the budding cycle. This structure participates in different aspects of morphogenesis, such as selection of cell polarity, localization of chitin synthesis, the switch from hyperpolar to isotropic bud growth after bud emergence and the spatial regulation of septation. The septin cytoskeleton assembles at the pre-bud site before bud emergence, remains there during bud growth and duplicates at late mitosis eventually disappearing after cell separation. Using a septin-GFP fusion and time-lapse confocal microscopy, we have determined that septin dynamics are maintained in budding zygotes and during unipolar synchronous growth in pseudohyphae. By means of specific cell cycle arrests and deregulation of cell cycle controls we show that septin assembly is dependent on G1 cy...
Septins may form a ubiquitous family of cytoskeletal filaments
Journal of Cell Biology, 1996
HIS minireview discusses recent information about the septin family of proteins, which suggests that the septins may be elements of a new filament system that functions in all or most eukaryotic cells. Septins are found in a wide variety of eukaryotes, are important for cytokinesis, and may compose or regulate a ubiquitous filament system that has not been previously recognized. A more extensive review of septins was recently published (26). This minireview also presents a new comparison of septin sequences. Actin filaments, microtubules, intermediate filaments, and myosin thick filaments, have been extensively studied over the past several decades, using methodologic advances in electron and light microscopy, detergent extraction of cells, and purification of proteins. This collection of filaments has been assumed to represent a fairly complete picture of the cytoskeletal filaments commonly found in eukaryotes. However, recent work suggests that cells augment these common cytoskeletal elements with additional elements, at least one of which, the septins, appears to be widely expressed and used for essential cell processes. Neck Filaments and Septins A set of ~10-nm filaments associated with the mother/bud neck of Saccharomyces cerevisiae, was discovered by thin section electron microscopy~(4, 5). These "neck filaments" are in a plane perpendicular to the mother/bud axis and are very near the plasma membrane. These properties are similar to those of the actin filaments in the contractile ring associated with cytokinesis in animal cells. However, the yeast neck filaments are observed during bud growth but not cytokinesis, unlike the actin filaments of the contractile ring of animal cells. Subsequent localization of actin in budding yeast confirmed the disparity between the neck filaments and actin, in that actin is present at the neck during bud emergence and cytokinesis, but not during bud growth, a temporal pattern clearly distinct from that of the neck filaments (1, 22). A family of proteins, termed septins, are essential for neck filament assembly and may indeed be the primary structural components of the neck filaments. Septins were discovered in S. cerevisiae through the analysis of cell divi-Please address all correspondence to J.
Cell integrity and morphogenesis in a budding yeast septin mutant
Microbiology, 1998
The non-sporulating diploid strain V327 of Saccharomyces cerevisiae was previously isolated in a search for thermosensitive autolytic mutants. This strain is very efficient a t releasing intracellular proteins into the medium when incubated a t high temperatures. The expression of this lytic phenotype depends on a morphogenetic defect, consisting of the appearance of elongated chains of cells. Transmission electron microscopy revealed a mislocalization of septa at semi-permissive temperatures and a total lack of septation together with abnormal cell wall architecture a t a non-permissive temperature. The septin-encoding CDClO gene was cloned by complementation of the pleiotropic phenotype of the V327 mutant. Rescue and sequencing of CDClO alleles from V327 revealed a point mutation that created a single amino acid change in a region which is well conserved among septins. This new allele was named cdcl0-77. The construction of a cdcl0-ll haploid strain by substituting the CDClO gene with the rescued allele permitted further genetic analyses of the mutation and allowed the construction of new homozygous cdcl0-ll diploid strains that showed a reduced ability to sporulate. Fusing both the wild-type and the cdcl0-ll alleles to green fluorescent protein (GFP) demonstrated that the mutation does not affect the localization of this septin to the bud neck a t the standard growth temperature of 24 OC, although the morphogenetic phenotype a t 37 OC parallels the disappearance of CdclO-GFP a t the ring encircling the septum area.