Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4

Abstract

Assembly at the mother–bud neck of a filamentous collar containing five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) is necessary for proper morphogenesis and cytokinesis. We show that Cdc10 and Cdc12 possess GTPase activity and appropriate mutations in conserved nucleotide-binding residues abrogate GTP binding and/or hydrolysis in vitro. In vivo, mutants unable to bind GTP prevent septin collar formation, whereas mutants that block GTP hydrolysis do not. GTP binding-defective Cdc10 and Cdc12 form soluble heteromeric complexes with other septins both in yeast and in bacteria; yet, unlike wild-type, mutant complexes do not bind GTP and do not assemble into filaments in vitro. Absence of a p21-activated protein kinase (Cla4) perturbs septin collar formation. This defect is greatly exacerbated when combined with GTP binding-defective septins; conversely, the septin collar assembly defect of such mutants is suppressed efficiently by CLA4 overexpression. Cla4 interacts directly with and phosphorylates certain septins in vitro and in vivo. Thus, septin collar formation may correspond to septin filament assembly, and requires both GTP binding and Cla4-mediated phosphorylation of septins.

Some assembly required: yeast septins provide the instruction manual

“…chitin synthases) and components of the bud-site-selection machinery (for an exhaustive catalog, see [14,27]). In many instances, the binding is direct [28]. A well-characterized example of this scaffold role occurs in the so-called morphogenesis checkpoint (Box 1).…”

“…The signature motifs (G-boxes) of the GTPase superfamily are present in all septin family members except ARTS, a splice variant of Sept4 that lacks the G4 box [56]. Many septins bind and hydrolyze GTP [7,28,45,55]. However, the rates of nucleotide exchange and hydrolysis observed in vitro are slow.…”

“…This intrinsic property seems unaffected by cellular factors because the turnover of GTP in septin complexes in vivo, judged by mass spectrometry, is also very slow, at least in budding yeast [38]. Using radioactively labeled nucleotides (or fluorescent or photo-activatable analogs), it has been shown that some septin subunits, for example yeast Cdc10 and Cdc12, and fly Pnut and Sep1, bind GTP whereas others, for example yeast Cdc3 and Cdc11, and fly Sep2, do not [7,28]. Nonetheless, mutating residues that should contribute to GTP binding in many septins, based on analogy to other GTPases, alters formation, appearance, localization and/or function of septin filaments [3,36,[57][58][59].…”

“…Nonetheless, mutating residues that should contribute to GTP binding in many septins, based on analogy to other GTPases, alters formation, appearance, localization and/or function of septin filaments [3,36,[57][58][59]. In budding yeast, mutations in the P-loop of Cdc10 and Cdc12 that prevent GTP binding in vitro, permit assembly of hetero-oligomeric septin complexes of normal composition, but prevent polymerization of these complexes into filaments and confer temperature-sensitive growth defects in vivo [28]. Preventing GTP binding to both Cdc10 and Cdc12 has a strongly synergistic effect and confers temperature-sensitive lethality.…”

“…Initially, a group who reported that addition of either GTP or GTPγS (but not GDP) promotes the polymerization of frog recombinant Sept2 suggested that this sort of model might apply to the assembly of septins into filaments [55]. However, no other published study shows that a single septin can polymerize; moreover, even when hetero-oligomeric septin complexes are used, regardless of species, no effect of exogenously added guanine nucleotide on the rate or extent of polymerization has been seen [28,45]. Because guanine nucleotides are present in purified complexes, but exogenously added guanine nucleotides have no ostensible effect on either the rate or the extent of assembly of these complexes into filaments in vitro, the tightly bound (non-exchangeable) guanine nucleotide that survives purification of the complex must be sufficient to promote polymerization.…”