A Saccharomyces cerevisiae autoselection system for optimised recombinant protein expression

Geymonat, Marco; Spanos, Adonis; Sedgwick, Steven G.

doi:10.1016/j.gene.2007.05.001

Cited by 28 publications

(28 citation statements)

References 40 publications

(48 reference statements)

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“…GST-tagged Rho3p and Rho4p were also produced in yeast to obtain modified GTPases and, in particular, prenylated Rho proteins. For this purpose, the coding sequences of the RHO3 and RHO4 genes were inserted into pMG1 (30). The recombinant p783 plasmid for the production of GST-tagged Rgd1p in bacteria (12) was used as template for inverse PCR for the construction of bacterial plasmids encoding various truncated forms of Rgd1p: Rgd1⌬1-300, Rgd1⌬1-350, Rgd1⌬1-450, Rgd1⌬301-350, and Rgd1⌬1-156.…”

Section: Methodsmentioning

confidence: 99%

Phosphoinositides Affect both the Cellular Distribution and Activity of the F-BAR-containing RhoGAP Rgd1p in Yeast

Prouzet‐Mauléon¹,

Lefebvre²,

Thoraval

et al. 2008

Journal of Biological Chemistry

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Cell polarity is a key element of development in most eukaryotes. The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p contains an F-BAR domain at its N-terminal end in addition to its RhoGAP domain at its C-terminal end. We demonstrate here that phospholipids discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity on Rho4p. The central region of the protein contiguous to the F-BAR domain is required for this stimulation. The F-BAR region binds to phosphoinositides in vitro and also plays a key role in the localization of Rgd1p to the bud tip and neck during the cell cycle. Studies of heat-sensitive mutants lacking phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate suggested that Rgd1p initially binds to Golgi membranes via phosphatidylinositol 4-phosphate and is then transported to the plasma membrane, where it binds phosphatidylinositol 4,5-biphosphate. We demonstrate here the dual effects of phosphoinositides on a RhoGTPase-activating protein. Phosphoinositides both regulate the recruitment and trafficking of Rgd1p to membranes via the F-BAR domain and specifically stimulate GTPase-activating protein activity, consistent with functional interplay between lipids, RhoGAP, and its related GTPases in yeast growth.

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Section: Methodsmentioning

confidence: 99%

Phosphoinositides Affect both the Cellular Distribution and Activity of the F-BAR-containing RhoGAP Rgd1p in Yeast

Prouzet‐Mauléon¹,

Lefebvre²,

Thoraval

et al. 2008

Journal of Biological Chemistry

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“…MBP-Bfa1 from Escherichia coli was purified as described previously (Geymonat et al 2007;Maekawa et al 2007). GST-Hof1 was purified from E. coli according to the manufacturer's instructions (GE Healthcare and EMD).…”

Section: Protein Purificationsmentioning

confidence: 99%

“…GST-Hof1 was purified from E. coli according to the manufacturer's instructions (GE Healthcare and EMD). Clb2-Cdk1, Dbf2-Mob1, and Cdc5 were purified from yeast cells as described (Ubersax et al 2003;Geymonat et al 2007). For mass spectrometric analysis, Hof1-3HA and Hof1-9Myc were purified from 1.6-L of culture of wild-type and grr1D cells as described in Immunoprecipitation Experiments, below.…”

Section: Protein Purificationsmentioning

confidence: 99%

Phosphorylation-dependent regulation of the F-BAR protein Hof1 during cytokinesis

Meitinger¹,

Boehm²,

Hofmann³

et al. 2011

Genes Dev.

177

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Spatial and timely coordination of cytokinesis is crucial for the maintenance of organelle inheritance and genome integrity. The mitotic exit network (MEN) pathway controls both the timely initiation of mitotic exit and cytokinesis in budding yeast. Here we identified the conserved F-BAR protein Hof1 as a substrate of the MEN kinase complex Dbf2-Mob1 during cytokinesis. We show that polo-like kinase Cdc5 first phosphorylates Hof1 to allow subsequent phosphorylation by Dbf2-Mob1. This releases Hof1 from the septin ring and facilitates Hof1 binding to the medial actomyosin ring (AMR), where Hof1 promotes AMR contraction and membrane ingression. Domain structure analysis established that the central, unstructured, region of Hof1, named the ring localization sequence (RLS), is sufficient to mediate Hof1's binding to the medial ring in a cell cycle-dependent manner. Genetic and functional data support a model in which Dbf2-Mob1 regulates Hof1 by inducing domain rearrangements, leading to the exposure of the Hof1 RLS domain during telophase.

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“…These allowed bacterial expression and purification using either the N-teminal 6His-tag or the GST-tag of the pET/pGEX vector. The plo1 + coding sequence was cloned by PCR into the budding-yeast expression vector pMH919 (Geymonat et al, 2007) to produce N-terminally tagged 6His-Plo1p.…”

Section: Dna Constructs Cdnas Of Fkh2mentioning

confidence: 99%

“…6His-Plo1p was expressed in budding yeast YAT287, by galactose-induced expression from the GAL1-10 promoter of the pMH919 expression vector (Geymonat et al, 2007), and purified using Zn-bound sepharose beads; its activity was confirmed by its ability to phosphorylate myelin basic protein (data not shown).…”

Section: Protein Overexpression and Purificationmentioning

confidence: 99%

Regulation of gene expression during M-G1-phase in fission yeast through Plo1p and forkhead transcription factors

Papadopoulou

Ohkura³

et al. 2008

Journal of Cell Science

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In fission yeast the expression of several genes during M-G1 phase is controlled by binding of the PCB binding factor (PBF) transcription factor complex to Pombe cell cycle box (PCB) promoter motifs. Three components of PBF have been identified, including two forkhead-like proteins Sep1p and Fkh2p, and a MADS-box-like protein, Mbx1p. Here, we examine how PBF is controlled and reveal a role for the Polo kinase Plo1p. plo1+ shows genetic interactions with sep1+, fkh2+ and mbx1+, and overexpression of a kinase-domain mutant of plo1 abolishes M-G1-phase transcription. Plo1p binds to and directly phosphorylates Mbx1p, the first time a Polo kinase has been shown to phosphorylate a MADS box protein in any organism. Fkh2p and Sep1p interact in vivo and in vitro, and Fkh2p, Sep1p and Plo1p contact PCB promoters in vivo. However, strikingly, both Fkh2p and Plo1p bind to PCB promoters only when PCB-controlled genes are not expressed during S- and G2-phase, whereas by contrast Sep1p contacts PCBs coincident with M-G1-phase transcription. Thus, Plo1p, Fkh2p and Sep1p control M-G1-phase gene transcription through a combination of phosphorylation and cell-cycle-specific DNA binding to PCBs.

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A Saccharomyces cerevisiae autoselection system for optimised recombinant protein expression

Cited by 28 publications

References 40 publications

Phosphoinositides Affect both the Cellular Distribution and Activity of the F-BAR-containing RhoGAP Rgd1p in Yeast

Phosphoinositides Affect both the Cellular Distribution and Activity of the F-BAR-containing RhoGAP Rgd1p in Yeast

Phosphorylation-dependent regulation of the F-BAR protein Hof1 during cytokinesis

Regulation of gene expression during M-G1-phase in fission yeast through Plo1p and forkhead transcription factors

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