Loss of CDC5 function in Saccharomyces cerevisiae leads to defects in Swe1p regulation and Bfa1p/Bub2p-independent cytokinesis

In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of budding yeast polo kinase Cdc5p, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal domain. Here we show that, at a semipermissive temperature, the cdc5-3 mutant exhibited a synergistic bud elongation and growth defect with loss of HSL1, a component important for normal G(2)/M transition. Loss of SWE1, which phosphorylates and inactivates the budding yeast Cdk1 homolog Cdc28p, suppressed the cdc5-3 hsl1Delta defect, suggesting that Cdc5p functions at a point upstream of Swe1p. In addition, the cdc5-4 and cdc5-7 mutants exhibited chained cell morphologies with shared cytoplasms between the connected cell bodies, indicating a cytokinetic defect. Close examination of these mutants revealed delayed septin assembly at the incipient bud site and loosely organized septin rings at the mother-bud neck. Components in the mitotic exit network (MEN) play important roles in normal cytokinesis. However, loss of BFA1 or BUB2, negative regulators of the MEN, failed to remedy the cytokinetic defect of these mutants, indicating that Cdc5p promotes cytokinesis independently of Bfa1p and Bub2p. Thus, Cdc5p contributes to the activation of the Swe1p-dependent Cdc28p/Clb pathway, normal septin function, and cytokinesis.     

Requirement for Bbp1p in the proper mitotic functions of Cdc5p in Saccharomyces cerevisiae

The polo-box domain of the budding yeast polo kinase Cdc5p plays an essential role for targeting the catalytic activity of Cdc5p to spindle pole bodies (SPBs) and cytokinetic neck-filaments. Here, we report the isolation of Bbp1p as a polo-box interacting protein by a yeast two-hybrid screen. Bbp1p localizes to the periphery of the central plaque of the SPB and plays an important role in SPB duplication. Similarly, Cdc5p localized to the cytoplasmic periphery of the SPB. In vitro binding studies showed that Cdc5p interacted with the N-terminal domain of Bbp1p (Bbp1pDeltaC), but apparently not with Mps2p, a component shown to form a stable complex with Bbp1p. In addition, Bbp1p, but likely not Mps2p, was required for proper localization of Cdc5p to the SPB. The C-terminal coiled-coil domain of Bbp1p (Bbp1p(243-385)), which is crucial for both the homodimerization and the SPB localization, could target the localization-defective Cdc5pDeltaC to the SPB and induce the release of Cdc14p from the nucleolus. Consistent with this observation, expression of CDC5DeltaC-BBP1(243-385) under CDC5 promoter control partially complemented the cdc5Delta defect. These data suggest that Bbp1pDeltaC interacts with the polo-box domain of Cdc5p, and this interaction is critical for the subcellular localization and mitotic functions of Cdc5p.     

The polo box is required for multiple functions of Plx1 in mitosis

Polo-like kinases comprise a family of evolutionarily conserved serine/threonine protein kinases that play multiple roles in cell cycle regulation. In addition to the N-terminal catalytic domain, polo-like kinases have one or two highly conserved C-terminal non-catalytic regions, termed polo boxes. These motifs are required for targeting these kinases to subcellular mitotic structures. Here we report that kinase-dead Xenopus polo-like kinase (Plx1NA) functions as a competitor of endogenous Plx1 for polo box binding site(s) and inhibits the activation of Cdc25C and the G(2)-M transition in vivo. However, kinase-dead Plx1 with a point mutation in the polo box region (Plx1NAWF) did not have inhibitory effects. The ability of Plx1NA to block activation of the anaphase-promoting complex/cyclosome also requires an intact polo box. Microinjection of Plx1NA but not Plx1NAWF mRNA into Xenopus embryos caused cleavage arrest and formation of monopolar spindles, an effect previously seen in embryos injected with anti-Plx1 antibody. Spindle assembly experiments in vitro also showed that only monopolar spindles formed in Xenopus egg extracts supplemented with recombinant Plx1NA and that the spindle assembly process was delayed. Taken together, these results indicate that the polo box is required for Plx1 function in both the G(2)-M and the metaphase/anaphase transitions during the cell cycle.     

Bni5p,a septin-interacting protein,is required for normal septin function and cytokinesis in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7Delta strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7Delta mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.     

Requirement for the budding yeast polo kinase Cdc5 in proper microtubule growth and dynamics

In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of the budding yeast polo kinase Cdc5, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal noncatalytic polo box domain, a region that is critical for proper subcellular localization. One of these mutants, cdc5-11, exhibited a temperature-sensitive growth defect with an abnormal spindle morphology. Strikingly, provision of a moderate level of benomyl, a microtubule-depolymerizing drug, permitted cdc5-11 cells to grow significantly better than the isogenic CDC5 wild type in a FEAR (cdc Fourteen Early Anaphase Release)-independent manner. In addition, cdc5-11 required MAD2 for both cell growth and the benomyl-remedial phenotype. These results suggest that cdc5-11 is defective in proper spindle function. Consistent with this view, cdc5-11 exhibited abnormal spindle morphology, shorter spindle length, and delayed microtubule regrowth at the nonpermissive temperature. Overexpression of CDC5 moderately rescued the spc98-2 growth defect. Interestingly, both Cdc28 and Cdc5 were required for the proper modification of the spindle pole body components Nud1, Slk19, and Stu2 in vivo. They also phosphorylated these three proteins in vitro. Taken together, these observations suggest that concerted action of Cdc28 and Cdc5 on Nud1, Slk19, and Stu2 is important for proper spindle functions.     

The Sak polo-box comprises a structural domain sufficient for mitotic subcellular localization

The small family of polo-like kinases (Plks) includes Cdc5 from Saccharomyces cerevisiae, Plo1 from Schizosaccharomyces pombe, Polo from Drosophila melanogaster and the four mammalian genes Plk1, Prk/Fnk, Snk and Sak. These kinases control cell cycle progression through the regulation of centrosome maturation and separation, mitotic entry, metaphase to anaphase transition, mitotic exit and cytokinesis. Plks are characterized by an N-terminal Ser/Thr protein kinase domain and the presence of one or two C-terminal regions of similarity, termed the polo box motifs. These motifs have been demonstrated for Cdc5 and Plk1 to be required for mitotic progression and for subcellular localization to mitotic structures. Here we report the 2.0 A crystal structure of a novel domain composed of the polo box motif of murine Sak. The structure consists of a dimeric fold with a deep interfacial cleft and pocket, suggestive of a ligand-binding site. We show that this domain forms homodimers both in vitro and in vivo, and localizes to centrosomes and the cleavage furrow during cytokinesis. The requirement of the polo domain for Plk family function and the unique physical properties of the domain identify it as an attractive target for inhibitor design.     

A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis

Coordination of mitotic exit with timely initiation of cytokinesis is critical to ensure completion of mitotic events before cell division. The Saccharomyces cerevisiae polo kinase Cdc5 functions in a pathway leading to the degradation of mitotic cyclin Clb2, thereby permitting mitotic exit. Here we provide evidence that Cdc5 also plays a role in regulating cytokinesis and that an intact polo-box, a conserved motif in the noncatalytic COOH-terminal domain of Cdc5, is required for this event. Depletion of Cdc5 function leads to an arrest in cytokinesis. Overexpression of the COOH-terminal domain of Cdc5 (cdc5DeltaN), but not the corresponding polo-box mutant, resulted in connected cells. These cells shared cytoplasms with incomplete septa, and possessed aberrant septin ring structures. Provision of additional copies of endogenous CDC5 remedied this phenotype, suggesting a dominant-negative inhibition of cytokinesis. The polo-box-dependent interactions between Cdc5 and septins (Cdc11 and Cdc12) and genetic interactions between the dominant-negative cdc5DeltaN and Cyk2/Hof1 or Myo1 suggest that direct interactions between cdc5DeltaN and septins resulted in inhibition of Cyk2/Hof1- and Myo1-mediated cytokinetic pathways. Thus, we propose that Cdc5 may coordinate mitotic exit with cytokinesis by participating in both anaphase promoting complex activation and a polo-box-dependent cytokinetic pathway.     

Modulation of septin higher-order structure by the Cdc28 protein kinase

Septins are a family of eukaryotic guanosine phosphate-binding proteins that form linear heterooligomeric complexes, which, in turn, polymerize end-on-end into filaments. These filaments further assemble into higher-order structures at distinct subcellular locations. Dynamic changes in the organization of septin cortex structures appear during cell cycle progression. A variety of regulatory proteins and posttranslational modifications are involved in changes to the structure of septin assemblies during the entire cell cycle. In particular, septin-associated protein kinases mediate changes to septin higher order structures or interconnect cellular morphogenesis with the cell cycle. Yeast cyclin-dependent kinase, a master cell cycle regulator, is required for the initiation of a new septin ring. Here, using epifluoresence and electron microscopy, we show that upon phosphorylation by the Cdc28 kinase, septin filaments disassemble into hetero-octameric building blocks, and that filament depolymerization is specifically G1 cyclin-dependent.     

The crystal structure of the human polo-like kinase-1 polo box domain and its phospho-peptide complex

Human polo-like kinase Plk1 localizes to the centrosomes, kinetochores and central spindle structures during mitosis. It plays an essential role in promoting mitosis and cytokinesis through phosphorylation of a number of different substrates. Kinase activity is regulated by a conserved C-terminal domain, termed the polo box domain (PBD), which acts both as an autoinhibitory domain and as a subcellular localization domain. We have determined the crystal structure of Plk1 PBD (residues 367-603) to 2.2 A resolution and the structure of a phospho-peptide-PBD (residues 345-603) complex to 2.3 A resolution. The two polo boxes of the PBD exhibit identical folds based on a six-stranded beta-sheet and an alpha-helix, despite only 12% sequence identity. The phospho-peptide binds at a site between the two polo boxes. It makes a short antiparallel beta-sheet connection and critical contacts to residues Trp414, Leu490, His538 and Lys540. Most of these residues had been shown to be important for biological activity through mutational studies. The results provide an explanation for phospho-peptide recognition and create the basis for new functional studies.     

Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function

Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.     

Kin2, the Budding Yeast Ortholog of Animal MARK/PAR-1 Kinases,Localizes to the Sites of Polarized Growth and May Regulate Septin Organization and the Cell Wall

MARK/PAR-1 protein kinases play important roles in cell polarization in animals. Kin1 and Kin2 are a pair of MARK/PAR-1 orthologs in the budding yeast Saccharomyces cerevisiae. They participate in the regulation of secretion and ER stress response. However, neither the subcellular localization of these two kinases nor whether they may have other cellular functions is clear. Here, we show that Kin2 localizes to the sites of polarized growth in addition to localization on the plasma membrane. The localization to polarity sites is mediated by two targeting domains—TD1 and TD2. TD1 locates in the N-terminal region that spans the protein kinase domain whereas TD2 locates in the C-terminal end that covers the KA1 domain. We also show that an excess of Kin2 activity impaired growth, septin organization, and chitin deposition in the cell wall. Both TD1 and TD2 contribute to this function. Moreover, we find that the C-terminal region of Kin2 interacts with Cdc11, a septin subunit, and Pea2, a component of the polarisome that is known to play a role in septin organization. These findings suggest that Kin2 may play a role in the regulation of the septin cytoskeleton and the cell wall. Finally, we show that the C-terminal region of Kin2 interacts with Rho3, a Rho GTPase, whereas the N-terminal region of Kin2 interacts with Bmh1, a 14-3-3 protein. We speculate that Kin2 may be regulated by Bmh1, Rho3, or Pea2 in vivo. Our study provides new insight in the localization, function, and regulation of Kin2.     

Hsl7 Localizes to a Septin Ring and Serves as an Adapter in a Regulatory Pathway That Relieves Tyrosine Phosphorylation of Cdc28 Protein Kinase in Saccharomyces cerevisiae

Successful mitosis requires faithful DNA replication, spindle assembly, chromosome segregation, and cell division. In the budding yeast Saccharomyces cerevisiae, the G(2)-to-M transition requires activation of Clb-bound forms of the protein kinase, Cdc28. These complexes are held in an inactive state via phosphorylation of Tyr19 in the ATP-binding loop of Cdc28 by the Swe1 protein kinase. The HSL1 and HSL7 gene products act as negative regulators of Swe1. Hsl1 is a large (1,518-residue) protein kinase with an N-terminal catalytic domain and a very long C-terminal extension. Hsl1 localizes to the incipient site of cytokinesis in the bud neck in a septin-dependent manner; however, the function of Hsl7 was not previously known. Using both indirect immunofluorescence with anti-Hsl7 antibodies and a fusion of Hsl7 to green fluorescent protein, we found that Hsl7 also localizes to the bud neck, congruent with the septin ring that faces the daughter cell. Both Swe1 and a segment of the C terminus of Hsl1 (which has no sequence counterpart in two Hsl1-related protein kinases, Gin4 and Kcc4) were identified as gene products that interact with Hsl7 in a two-hybrid screen of a random S. cerevisiae cDNA library. Hsl7 plus Swe1 and Hsl7 plus Hsl1 can be coimmunoprecipitated from extracts of cells overexpressing these proteins, confirming that Hsl7 physically associates with both partners. Also consistent with the two-hybrid results, Hsl7 coimmunoprecipitates with full-length Hsl1 less efficiently than with a C-terminal fragment of Hsl1. Moreover, Hsl7 does not localize to the bud neck in an hsl1Delta mutant, whereas Hsl1 is localized normally in an hsl7Delta mutant. Phosphorylation and ubiquitinylation of Swe1, preludes to its destruction, are severely reduced in cells lacking either Hsl1 or Hsl7 (or both), as judged by an electrophoretic mobility shift assay. Collectively, these data suggest that formation of the septin rings provides sites for docking Hsl1, exposing its C terminus and thereby permitting recruitment of Hsl7. Hsl7, in turn, presents its cargo of bound Swe1, allowing phosphorylation by Hsl1. Thus, Hsl1 and Hsl7 promote proper timing of cell cycle progression by coupling septin ring assembly to alleviation of Swe1-dependent inhibition of Cdc28. Furthermore, like septins and Hsl1, homologs of Hsl7 are found in fission yeast, flies, worms, and humans, suggesting that its function in this control mechanism may be conserved in all eukaryotes.     

Late mitotic failure in mice lacking Sak, a polo-like kinase

Polo-like kinases in yeast, flies, and mammals regulate key events in mitosis. Such events include spindle formation at G2/M, the anaphase-promoting complex (APC) at the exit from mitosis, the cleavage structure at cytokinesis, and DNA damage checkpoints in G2/M. Polo-like kinases are distinguished by two C-terminal polo box (pb) motifs, which localize the enzymes to mitotic structures. We previously identified Sak, a novel polo-like kinase found in Drosophila and mammals. Here, we demonstrate that the Sak kinase has a functional pb domain that localizes the enzyme to the nucleolus during G2, to the centrosomes in G2/M, and to the cleavage furrow during cytokinesis. To study the role of Sak in embryo development, we generated a Sak null allele, the first polo-like kinase to be mutated in mice. Sak(-/-) embryos arrested after gastrulation at E7.5, with a marked increase in mitotic and apoptotic cells. Sak(-/-) embryos displayed cells in late anaphase or telophase that continued to express cyclin B1 and phosphorylated histone H3. Our results suggest that Sak is required for the APC-dependent destruction of cyclin B1 and for exit from mitosis in the postgastrulation embryo.     

Protein-protein interactions governing septin heteropentamer assembly and septin filament organization in Saccharomyces cerevisiae

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.     

Structure and substrate recruitment of the human spindle checkpoint kinase Bub1

In mitosis, the spindle checkpoint detects a single unattached kinetochore, inhibits the anaphase-promoting complex or cyclosome (APC/C), and prevents premature sister chromatid separation. The checkpoint kinase Bub1 contributes to checkpoint sensitivity through phosphorylating the APC/C activator, Cdc20, and inhibiting APC/C catalytically. We report here the crystal structure of the kinase domain of Bub1, revealing the requirement of an N-terminal extension for its kinase activity. Though the activation segment of Bub1 is ordered and has structural features indicative of active kinases, the C-terminal portion of this segment sterically restricts substrate access to the active site. Bub1 uses docking motifs, so-called KEN boxes, outside its kinase domain to recruit Cdc20, one of two known KEN box receptors. The KEN boxes of Bub1 are required for the spindle checkpoint in human cells. Therefore, its unusual active-site conformation and mode of substrate recruitment suggest that Bub1 has an exquisitely tuned specificity for Cdc20.     

Polo-like kinase 1 in the life and death of cancer cells

The polo-like kinase family plays a vital role in many cell cycle related events. The family includes mammalian Plkl, Snk (Plk2), and Fnk/Prk (Plk3), Xenopus laevis Plxl,Drosophila polo, fission yeast Plol, and budding yeast Cdc5. These enzymes, in addition to a conserved kinase domain at the N-terminus, have highly conserved sequences called polo-box(s) in the non-catalytic C-terminal domain. Genetic and biochemical experiments with several different organisms have documented that polo-like kinases are involved in many aspects of the cell cycle, such as activation of Cdc2, centrosome assembly and maturation, activation of the anaphase-promoting complex (APC) during the metaphase-anaphase transition, and cytokinesis.     

Polo-Like Kinase 1 in the Life and Death of Cancer Cells

The polo-like kinase family plays a vital role in many cell cycle related events. The family includes mammalian Plk1, Snk (Plk2), and Fnk/Prk (Plk3), Xenopus laevis Plx1, Drosophila polo, fission yeast Plo1, and budding yeast Cdc5. These enzymes, in addition to a conserved kinase domain at the N-terminus, have highly conserved sequences called polo-box(s) in the non-catalytic C-terminal domain.1 Genetic and biochemical experiments with several different organisms have documented that polo-like kinases are involved in many aspects of the cell cycle, such as activation of Cdc2, centrosome assembly and maturation, activation of the anaphase-promoting complex (APC) during the metaphase-anaphase transition, and cytokinesis.(1-3)     

The germinal centre kinase Don3 triggers the dynamic rearrangement of higher‐order septin structures during cytokinesis in Ustilago maydis

The dimorphic phytopathogenic fungus Ustilago maydis grows in its haploid phase by budding. Cytokinesis and separation of daughter cells are accomplished by the consecutive formation of two distinct septa. Here, we show that both septation events involve the dynamic rearrangement of septin assemblies from hourglass‐shaped collars into ring‐like structures. Using a chemical genetic approach we demonstrate that the germinal centre kinase Don3 triggers this septin reorganization during secondary septum formation. Although chemical inhibition of an analogue‐sensitive version of Don3 prevented septation, a stable septin collar was assembled at the presumptive septation site. Interestingly, the essential light chain of type II myosin, Cdc4, was already associated with this septin collar. Release of Don3 kinase inhibition triggered immediate dispersal of septin filaments and concomitant incorporation of Cdc4 into a contractile actomyosin ring, which also contained the F‐BAR domain protein Cdc15. Inhibition of actin polymerization or deletion of the cdc15 gene, did not affect assembly of the initial collar consisting of septin and myosin light chain. However, reassembly of septin filaments into a ring‐like structure was prevented in the absence of either F‐actin or Cdc15, indicating that septin ring formation in U. maydis depends on a functional contractile actomyosin ring.     

Dictyostelium Aurora kinase has properties of both Aurora A and Aurora B kinases

Aurora kinases are highly conserved proteins with important roles in mitosis. Metazoans contain two kinases, Aurora A and B, which contribute distinct functions at the spindle poles and the equatorial region respectively. It is not currently known whether the specialized functions of the two kinases arose after their duplication in animal cells or were already present in their ancestral kinase. We show that Dictyostelium discoideum contains a single Aurora kinase, DdAurora, that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box sequence and localizes at the spindle poles during early mitosis. Like Aurora B, DdAurora binds to its partner DdINCENP and localizes on centromeres at metaphase, the central spindle during anaphase, and the cleavage furrow at the end of cytokinesis. DdAurora also has several unusual properties. DdAurora remains associated with centromeres in anaphase, and this association does not require an interaction with DdINCENP. DdAurora then localizes at the cleavage furrow, but only at the end of cytokinesis. This localization is dependent on DdINCENP and the motor proteins Kif12 and myosin II. Thus, DdAurora may represent the ancestral kinase that gave rise to the different Aurora kinases in animals and also those in other organisms.     

Interplay of septin amphipathic helices in sensing membrane-curvature and filament bundling

The curvature of the membrane defines cell shape. Septins are GTP-binding proteins that assemble into heteromeric complexes and polymerize into filaments at areas of micron-scale membrane curvature. An amphipathic helix (AH) domain within the septin complex is necessary and sufficient for septins to preferentially assemble onto micron-scale curvature. Here we report that the nonessential fungal septin, Shs1, also has an AH domain capable of recognizing membrane curvature. In a septin mutant strain lacking a fully functional Cdc12 AH domain (cdc12-6), the C-terminal extension of Shs1, containing an AH domain, becomes essential. Additionally, we find that the Cdc12 AH domain is important for regulating septin filament bundling, suggesting septin AH domains have multiple, distinct functions and that bundling and membrane binding may be coordinately controlled.