Multiple Functional Domains of the Yeast l,3-β-Glucan Synthase Subunit Fks1p Revealed by Quantitative Phenotypic Analysis of Temperature-Sensitive Mutants

Okada, Hiroki; Abe, Mitsuhiro; Asakawa-Minemura, Masayo; Hirata, Aiko; Qadota, Hiroshi; Morishita, K.; Ohnuki, Shinsuke; Nogami, Satoru; Ohya, Yoshikazu

doi:10.1534/genetics.109.109892

Cited by 57 publications

(56 citation statements)

References 53 publications

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“…The temperature-sensitive FKS1 mutant (catalytic subunit of 1,3-␤-glucan synthase [32]) fks1-1154 loses its synthase activity at the restrictive temperature of 37°C due to an impaired supply of cell wall material to growing buds (33). As a consequence, fks1-1154 cells undergo budding cycle arrest with no or small-budded cells and, more interestingly, with duplicated and adjacent SPBs and replicated DNA, which is described as the cell wall integrity cell cycle checkpoint (3).…”

Section: Resultsmentioning

confidence: 99%

The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint

Negishi

Veis

Hollenstein

et al. 2016

Molecular and Cellular Biology

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The cell wall integrity (CWI) checkpoint in the budding yeast Saccharomyces cerevisiae coordinates cell wall construction and cell cycle progression. In this study, we showed that the regulation of Hcm1, a late-S-phase transcription factor, arrests the cell cycle via the cell wall integrity checkpoint. Although the HCM1 mRNA level remained unaffected when the cell wall integrity checkpoint was induced, the protein level decreased. The overproduction of Hcm1 resulted in the failure of the cell wall integrity checkpoint. We identified 39 Hcm1 phosphorylation sites, including 26 novel sites, by tandem mass spectrometry analysis. A mutational analysis revealed that phosphorylation of Hcm1 at S61, S65, and S66 is required for the proper onset of the cell wall integrity checkpoint by regulating the timely decrease in its protein level. Hyperactivation of the CWI mitogen-activated protein kinase (MAPK) signaling pathway significantly reduced the Hcm1 protein level, and the deletion of CWI MAPK Slt2 resulted in a failure to decrease Hcm1 protein levels in response to stress, suggesting that phosphorylation is regulated by CWI MAPK. In conclusion, we suggest that Hcm1 is regulated negatively by the cell wall integrity checkpoint through timely phosphorylation and degradation under stress to properly control budding yeast proliferation. Critical events during cell cycle progression, such as DNA replication and mitosis, are regulated by cell cycle checkpoints to ensure the proper completion of cell division. The cell cycle in the budding yeast Saccharomyces cerevisiae is coordinated with bud growth to secure the morphological space for cell division (1). One of the cell cycle checkpoints required to monitor budding morphology is the morphogenesis checkpoint (2), and the other is the cell wall integrity (CWI) checkpoint (3). They function independently to coordinate bud growth and cell wall construction, respectively, with cell cycle progression (4). Without a supply of cell wall materials for bud growth, the budding yeast cell cycle is arrested before entry into M phase and cannot proceed to mitosis (3).Compared to the morphogenesis checkpoint that regulates Swe1 phosphorylation and its degradation to activate cyclin-dependent kinase (CDK) (5), the primary function of the cell wall integrity checkpoint is inhibiting cell cycle progression to mitosis, which is achieved essentially by downregulating M-phase cyclin CLB2 (3, 4). During functions of the active cell wall integrity checkpoint, cells accumulate at the small budded state. Budding starts as cells proceed to S phase; thus, the checkpoint must be activated during S phase to prevent later cell cycle events. However, the manner by which the regulatory signaling cascade induced by the cell wall integrity checkpoint feeds into the complex transcriptional network of cell cycle progression has not been fully investigated. Genome-wide transcriptional studies have revealed the transcription cascade of cell cycle-related transcription factors (6-9). Studies have shown major tr...

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Section: Resultsmentioning

confidence: 99%

The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint

Negishi

Veis

Hollenstein

et al. 2016

Molecular and Cellular Biology

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“…Although the enzyme has not been purified to homogeneity, the central domain of partially purified Neurospora crassa Fks protein was shown to crosslink to azido-UDP-glucose (Schimoler-O' Rourke et al 2003), supporting the conclusion that this protein is the catalytic subunit. A recent functional analysis of FKS1 revealed that mutations in this central domain, which is predicted to be cytoplasmic, cause defects in GS activity (Okada et al 2010). Unlike loss of Pkc1, loss of Fks1/2 is not suppressed by increased osmotic support.…”

Section: Pkc1 and The Cwi Mapk Cascadementioning

confidence: 99%

Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

2011

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The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to the Rho1 GTPase, which mobilizes a physiologic response through a variety of effectors. Activation of CWI signaling regulates the production of various carbohydrate polymers of the cell wall, as well as their polarized delivery to the site of cell wall remodeling. This review article centers on CWI signaling in Saccharomyces cerevisiae through the cell cycle and in response to cell wall stress. The interface of this signaling pathway with other pathways that contribute to the maintenance of cell wall integrity is also discussed.

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“…A similar attempt to map the binding site for the substrate UDP-glucose using partially purified ␤-1,3-glucan synthase from Neurospora crassa also suffered from low specificity; however, among the cross-linked peptides were four clustered between residues equivalent to 1070 to 1273 in S. cerevisiae Fks1 (41). More recently, the characterization of temperature-sensitive S. cerevisiae mutants implicated specific Fks1 residues in ␤-1,3-glu-can synthase activity, as well as other phenotypes, including polarized growth and endocytosis (33).…”

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confidence: 99%

Topological and Mutational Analysis of Saccharomyces cerevisiae Fks1

Johnson

Edlind

2012

Eukaryot Cell

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ABSTRACTFks1, with orthologs in nearly all fungi as well as plants and many protists, plays a central role in fungal cell wall formation as the putative catalytic component of β-1,3-glucan synthase. It is also the target for an important new antifungal group, the echinocandins, as evidenced by the localization of resistance-conferring mutations to Fks1 hot spots 1, 2, and 3 (residues 635 to 649, 1354 to 1361, and 690 to 700, respectively). Since Fks1 is an integral membrane protein and echinocandins are cyclic peptides with lipid tails, Fks1 topology is key to understanding its function and interaction with echinocandins. We used hemagglutinin (HA)-Suc2-His4C fusions to C-terminally truncatedSaccharomyces cerevisiaeFks1 to experimentally define its topology and site-directed mutagenesis to test function of selected residues. Of the 15 to 18 transmembrane helices predictedin silicofor Fks1 from evolutionarily diverse fungi, 13 were experimentally confirmed. The N terminus (residues 1 to 445) is cytosolic and the C terminus (residues 1823 to 1876) external; both are essential to Fks1 function. The cytosolic central domain (residues 715 to 1294) includes newly recognized homology to glycosyltransferases, and residues potentially involved in substrate UDP-glucose binding and catalysis are essential. All three hot spots are external, with hot spot 1 adjacent to and hot spot 3 largely embedded within the outer leaflet of the membrane. This topology suggests a model in which echinocandins interact through their lipid tails with hot spot 3 and through their cyclic peptides with hot spots 1 and 2.

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Multiple Functional Domains of the Yeast l,3-β-Glucan Synthase Subunit Fks1p Revealed by Quantitative Phenotypic Analysis of Temperature-Sensitive Mutants

Cited by 57 publications

References 53 publications

The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint

The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint

Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

Topological and Mutational Analysis of Saccharomyces cerevisiae Fks1

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