Shs1p: A Novel Member of Septin That Interacts with Spa2p, Involved in Polarized Growth inSaccharomyces cerevisiae

Mino, Akihisa; Tanaka, Kazuma; Kamei, Takashi; Umikawa, Masato; Fujiwara, Takeshi; Takai, Yoshimi

doi:10.1006/bbrc.1998.9541

Cited by 94 publications

(100 citation statements)

References 34 publications

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“…Twenty-two of the candidate substrates we identified have known cellular localizations. Five of them (Shs1, Pam1, Kcc4, Rpg1, and Rom2) (Ϸ23%) are found at the bud or bud neck (28)(29)(30)(31)(32), Ͼ7-fold more than would be expected by random sampling. Among the five bud neck and bud localized substrates are three of the six Pcl1-specific substrates, including a structural component of the septin ring and known regulators of septin dynamics and actin polarization.…”

Section: Resultsmentioning

confidence: 94%

Combining chemical genetics and proteomics to identify protein kinase substrates

Dephoure

Howson²,

Blethrow³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

111

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Phosphorylation is a ubiquitous protein modification important for regulating nearly every aspect of cellular biology. Protein kinases are highly conserved and constitute one of the largest gene families. Identifying the substrates of a kinase is essential for understanding its cellular role, but doing so remains a difficult task. We have developed a high-throughput method to identify substrates of yeast protein kinases that employs a collection of yeast strains each expressing a single epitope-tagged protein and a chemical genetic strategy that permits kinase reactions to be performed in native, whole-cell extracts. Using this method, we screened 4,250 strains expressing epitope-tagged proteins and identified 24 candidate substrates of the Pho85-Pcl1 cyclin-dependent kinase, including the known substrate Rvs167. The power of this method to identify true kinase substrates is strongly supported by functional overlap and colocalization of candidate substrates and the kinase, as well as by the specificity of Pho85-Pcl1 for some of the substrates compared with another Pho85-cyclin kinase complex. This method is readily adaptable to other yeast kinases.cell signaling ͉ phosphorylation ͉ cyclin-dependent kinase T he general criteria for establishing that a protein is a substrate of a given kinase are the ability of the kinase to phosphorylate the substrate in vitro and the dependence on the activity of the kinase for phosphorylation of the substrate in vivo (1). In vivo validation of kinase substrates continues to be laborious and time-consuming. Thus, it is crucial to be able to efficiently identify candidates before committing to this step. Indirect data, such as genetic and physical interactions, can provide insights into potential substrates, but many interacting proteins and genes are not substrates (1, 2). More direct approaches using in vitro kinase reactions have been attempted in many permutations (1, 2). Purified component reactions directly measure the ability of a kinase to phosphorylate a particular substrate and can be scaled for high-throughput formats, but such conditions often compromise reaction specificity and produce false-positive results (1, 2).We sought to improve on these techniques by carrying out a biochemical screen in an environment that more closely resembles the in vivo state. To do so, we carried out kinase reactions with near physiological levels of exogenous kinase in native whole-cell extracts. These extracts maintain protein-protein interactions that may affect substrate presentation and preserve a nearly full complement of cellular proteins, including potential adaptor proteins and cofactors that could affect the reaction. These reaction conditions also preserve the native relative protein abundances and a natural competition among substrates for limited kinase.A challenge of carrying out protein kinase reactions in wholecell lysates is that one cannot attribute phosphorylation of a protein to a particular kinase in the reaction, because all kinases in the extract are capable of cat...

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Section: Resultsmentioning

confidence: 94%

Combining chemical genetics and proteomics to identify protein kinase substrates

Dephoure

Howson²,

Blethrow³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

111

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“…However, our observations that Shs1 overproduction did not suppress temperature sensitivity of the cdc11-1 mutant (our unpublished data) and that overproduction of Shs1 was toxic to the cdc11D cells ( Figure 5D) indicate that Shs1 and Cdc11 are not functionally interchangeable. These seemingly conflicting genetic observations can be explained by assuming that Shs1, but not Cdc11, is able to interact with a polarity or bud-cortex localized protein, such as Spa2 (Mino et al 1998). This assumption is supported by the septin localization data presented in Figure 5C.…”

Section: Shs1 Acts Differently In Cytokinesis From Other Septinsmentioning

confidence: 91%

“…The amino acid sequences of the gene products deduced from the nucleotide sequences of these genes are similar to each other, and these proteins are collectively designated septins (Haarer and Pringle 1987;Ford and Pringle 1991;Kim et al 1991;Sanders and Field 1994). Two other septins, Spr3 and Spr28, are spore specific, and a seventh septin, Shs1/Sep7, was identified on the basis of sequence homology (Ozsarac et al 1995;De Virgilio et al 1996;Carroll et al 1998;Mino et al 1998).…”

mentioning

confidence: 96%

Shs1 Plays Separable Roles in Septin Organization and Cytokinesis in Saccharomyces cerevisiae

Iwase

Luo

et al. 2007

Genetics

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In Saccharomyces cerevisiae, five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7) form the septin ring at the bud neck during vegetative growth. We show here that disruption of SHS1 caused cold-sensitive growth in the W303 background, with cells arrested in chains, indicative of a cytokinesis defect. Surprisingly, the other four septins appeared to form an apparently normal septin ring in shs1D cells grown under the restrictive condition. We found that Myo1 and Iqg1, two components of the actomyosin contractile ring, and Cyk3, a component of the septum formation, were either delocalized or mislocalized in shs1D cells, suggesting that Shs1 plays supportive roles in cytokinesis. We also found that deletion of SHS1 enhanced or suppressed the septin defect in cdc10D and cdc11D cells, respectively, suggesting that Shs1 is involved in septin organization, exerting different effects on septin-ring assembly, depending on the composition of the septin subunits. Furthermore, we constructed an shs1-100c allele that lacks the coding sequence for the C-terminal 32 amino acids. This allele still displayed the genetic interactions with the septin mutants, but did not show cytokinesis defects as described above, suggesting that the roles of Shs1 in septin organization and cytokinesis are separable.

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“…In Saccharomyces cerevisiae, there are seven septin genes, of which five (CDC3, CDC10, CDC11, CDC12, and SHS1/SEP7) are expressed in vegetative cells (Longtine et al, 1996;Carroll et al, 1998;Mino et al, 1998) and two (SPR3 and SPR28) only during sporulation (DeVirgilio et al, 1996;Fares et al, 1996). The five vegetatively expressed septins have identical localization patterns throughout the cell cycle.…”

Section: Introductionmentioning

confidence: 99%

“…In each species examined to date, there are two or more septins that form heterooligomeric complexes to perform their specific functions. Purified septins from yeast, Drosophila, Xenopus, and mammalian cells form filaments of 7-9 nm diameter and of variable length (Field et al, 1996;Frazier et al, 1998;Hsu et al, 1998;Kinoshita et al, 2002;Mendoza et al, 2002).In Saccharomyces cerevisiae, there are seven septin genes, of which five (CDC3, CDC10, CDC11, CDC12, and SHS1/SEP7) are expressed in vegetative cells (Longtine et al, 1996;Carroll et al, 1998;Mino et al, 1998) and two (SPR3 and SPR28) only during sporulation (DeVirgilio et al, 1996;Fares et al, 1996). The five vegetatively expressed septins have identical localization patterns throughout the cell cycle.…”

mentioning

confidence: 99%

The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

Caviston

Longtine

Pringle

et al. 2003

MBoC

226

281

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The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42 V36G allele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42 V36G or GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth. INTRODUCTIONThe septin family of proteins was first recognized in yeast and now appears to be ubiquitous in fungi and animals, although not in plants (Longtine et al., 1996;Field and Kellogg, 1999;Trimble, 1999;Nguyen et al., 2000;Momany et al., 2001;Macara et al., 2002). Typical septins have a variable N-terminal region, a conserved core that includes the elements of a GTP-binding site, and a variable C-terminal region that (in all but a few cases) includes a predicted coiled-coil domain. In each species examined to date, there are two or more septins that form heterooligomeric complexes to perform their specific functions. Purified septins from yeast, Drosophila, Xenopus, and mammalian cells form filaments of 7-9 nm diameter and of variable length (Field et al., 1996;Frazier et al., 1998;Hsu et al...

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Shs1p: A Novel Member of Septin That Interacts with Spa2p, Involved in Polarized Growth inSaccharomyces cerevisiae

Cited by 94 publications

References 34 publications

Combining chemical genetics and proteomics to identify protein kinase substrates

Combining chemical genetics and proteomics to identify protein kinase substrates

Shs1 Plays Separable Roles in Septin Organization and Cytokinesis in Saccharomyces cerevisiae

The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

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