Requirements of Fission Yeast Septins for Complex Formation, Localization, and Function

Abstract

Septins are GTP binding proteins important for cytokinesis in many eukaryotes. The Schizosaccaromyces pombe genome sequence predicts orthologues of four of five Saccharomyces cerevisiae septins involved in cytokinesis and these are named Spns1-4p. That spns1-4 are not essential genes permitted the application of a combined genetic and proteomics approach to determine their functional relationships. Our findings indicate that Spns1-4p are present throughout interphase as a diffusely localized approximately 8.5S complex containing two copies of each septin linked together as a chain in the order Spn3p-Spn4p-Spn1p-Spn2p. Septin recruitment to the medial region of the cell is genetically separable from ring formation, and whereas it is normally restricted to mitosis, it can be promoted without activation of the mitotic cell cycle machinery. Coalescence into ring structures requires Spn1p and Spn4p associate with at least one other septin subunit and the expression of Mid2p that is normally restricted to mitosis. This study establishes the functional requirements for septin complex organization in vivo.

Some assembly required: yeast septins provide the instruction manual

“…The amounts of the various subunits present in and the sedimentation coefficient of the complex are, again, consistent with two copies of each septin in a 1:1:1:1 stoichiometry. Moreover in agreement with the view that the Spn1 (Cdc3)-Spn4 (Cdc12) heterotetramer forms the core of the complex, deletion of either of these subunits causes a much more severe phenotype (cell-separation defect) than absence of either Spn2 (Cdc10) or Spn3 (Cdc11) [33].…”

“…During cytokinesis, the tubular collar of septin filaments is split into two separate 'rings' that seem to serve as gaskets that confine and concentrate between them factors that are involved directly in cytokinesis, such as the exocyst component Sec3, an adaptor protein Spa2 and an isoform of chitin synthase Chs2 [31]. This compartmentalization function appears to be conserved in fission yeast, where a double ring of septin filaments encompasses the contractile apparatus and provides for efficient dissolution of the primary septum [32,33]. It appears that septins might have a similar role in mammalian cells because septins are prominent components of the cleavage furrow and a diffusion barrier within the cleavage furrow has been demonstrated [34].…”

“…Although Spn5, Spn6 and Spn7 from S. pombe have not been studied in detail, they probably represent sporulation-specific septins [43]. The structure of the septin complex in mitotic cells in fission yeast has been analyzed in detail [33], and is similar to that of budding yeast (Figure 1b). Spn1 (Cdc3) binds to Spn4 (Cdc12), and this complex is, in turn, stabilized by Spn2 (Cdc10).…”

“…With the notable exception of Xenopus laevis Sept2 [55], single septins and complexes that are composed of two different septins are not reported to polymerize in vitro. In all other cases examined, the capacity to polymerize into filaments requires the presence of complexes that contain at least three types of septin, for example, Cdc3-Cdc12-Cdc11 in budding yeast [10], Spn1-Spn4-Spn3 and Spn1-Spn4-Spn2 in fission yeast [33], Sept6-Sept7-Sept2 in mammalian cells [9,45] and Sep2-Pnut-Sep1 in flies [7]. Although the Cdc3-Cdc12-Cdc11-containing complex polymerizes into filaments in vitro, Cdc10 makes crucial contributions to septin-filament organization and stability in budding yeast (Box 2) [10], and Cdc10 counterparts in other organisms presumably serve similar functions.…”

“…Indeed, ectopic expression of a stable, truncated derivative of Mid2 (endogenous expression of Mid2 is restricted to anaphase) leads to persistent septin rings, presumably because disassembly is impeded. Although septins are recruited to the medial cortex of fission yeast cells in the absence of Mid2 (possibly by association with membrane phosphoinositides), they do not coalesce into a proper ring structure that, presumably, corresponds to authentic filament formation [33]. The closest counterpart of Mid2 in budding yeast appears to be Bud4, which localizes to the bud neck and associates with other markers of the bud neck, such as IQGAP (Iqg1) [78], which, in fission yeast, are also recruited early to the medial cytokinetic ring [32].…”