Three-dimensional ultrastructure of the septin filament network inSaccharomyces cerevisiae (original) (raw)
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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.
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.
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 ...
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-dependent compartmentalization of the endoplasmic reticulum during yeast polarized growth
Journal of Cell Biology, 2005
olarized cells frequently use diffusion barriers to separate plasma membrane domains. It is unknown whether diffusion barriers also compartmentalize intracellular organelles. We used photobleaching techniques to characterize protein diffusion in the yeast endoplasmic reticulum (ER). Although a soluble protein diffused rapidly throughout the ER lumen, diffusion of ER membrane proteins was restricted at the bud neck. Ultrastructural studies and fluorescence microscopy revealed the presence of a ring of smooth ER at the bud neck. This ER domain P and the restriction of diffusion for ER membrane proteins through the bud neck depended on septin function. The membrane-associated protein Bud6 localized to the bud neck in a septin-dependent manner and was required to restrict the diffusion of ER membrane proteins. Our results indicate that Bud6 acts downstream of septins to assemble a fence in the ER membrane at the bud neck. Thus, in polarized yeast cells, diffusion barriers compartmentalize the ER and the plasma membrane along parallel lines.
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.