Protein–Protein Interactions Governing Septin Heteropentamer Assembly and Septin Filament Organization inSaccharomyces cerevisiae

Abstract

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.

Some assembly required: yeast septins provide the instruction manual

“…In addition, each septin (Cdc3, Cdc10, Cdc11 and Cdc12) can self-associate. In this regard, the apparent molecular weight of recombinant complexes isolated at a moderate salt concentration (0.25 M) is consistent with two copies of each septin in a 1:1:1:1 stoichiometry [10], whereas the amount of Cdc11 in native complexes that are isolated at higher salt concentrations (0.5-1.0 M) is substoichiometric [8,38]. Likewise, in high-salt conditions, the amount of Shs1 recovered in native complexes is substoichiometric [39].…”

“…Finally, a region at or near the C-terminus of most, but not all, septins is predicted to form a coiled coil because of the presence of an extended string of hydrophobic residues in the characteristic (but occasionally interrupted) 4-3 repeating pattern. Synthetic peptides that correspond to the predicted coiled-coils alone do not adopt a stable helical structure, either by themselves orwhenmixed withputative partner coiled-coil peptides from other septins [10]. A predicted α-helical region that precedes the coiled-coil segment appears to promote and stabilize coiled-coil formation, because a fragment of the C-terminus of Cdc12 that contains both the predicted α-helix and the coiled-coil sequence heterodimerizes with the C-terminus of Cdc3 [10].…”

“…Synthetic peptides that correspond to the predicted coiled-coils alone do not adopt a stable helical structure, either by themselves orwhenmixed withputative partner coiled-coil peptides from other septins [10]. A predicted α-helical region that precedes the coiled-coil segment appears to promote and stabilize coiled-coil formation, because a fragment of the C-terminus of Cdc12 that contains both the predicted α-helix and the coiled-coil sequence heterodimerizes with the C-terminus of Cdc3 [10]. Hereafter, we refer to the region that contains the predicted α-helix and the coiledcoil element as the C-terminal extension (CTE) (Figure 1a).…”

“…The contacts that mediate the interactions between the mitotic septins of S. cerevisiae have been mapped in detail [10]. Cdc3 and Cdc12 associate via their CTEs, and the CTEs are sufficient for this interaction.…”

“…Septin monomers assemble into hetero-oligomeric (multi-septin) complexes (models based on the available evidence are shown in Figure 1b). The complexes polymerize into filaments in vitro (Figure 2a) [7][8][9][10] and in vivo (Figure 2b) [11][12][13]. A model of filaments in S. cerevisiae is shown in Figure 2c.…”