Use of conditional promoters for expression of heterologous proteins in Saccharomyces cerevisiae

Rönicke, Volker; Graulich, Wolff; Mumberg, Dominik; Müller, Rolf; Funk, Martin

doi:10.1016/s0076-6879(97)83025-x

Cited by 34 publications

(25 citation statements)

References 13 publications

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“…We expressed ENTH1 or ENTH2 in W303 cells from a methioninerepressible MET25 promoter in a high-copy (2 μ) plasmid (Rönicke et al, 1997;Sikorski and Hieter, 1989). Under overexpression conditions (no methionine for 36 hours), ENTH2 led to significantly reduced growth compared to cells overexpressing ENTH1 or bearing empty vector (Fig.…”

Section: Overexpression Of Enth2 In Yeast Cells Induces Defects In Cementioning

confidence: 99%

The yeast endocytic protein Epsin 2 functions in a cell-division signaling pathway

Mukherjee

Coon

Edwards

et al. 2009

Journal of Cell Science

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The epsins are a family of adaptors involved in recruiting other endocytic proteins, binding of ubiquitylated cargo and induction of membrane curvature. These molecules bear a characteristic epsin N-terminal homology (ENTH) domain and multiple peptide motifs that mediate protein-protein interactions. We have previously demonstrated that the ENTH domain of epsin is involved in Cdc42 signaling regulation. Here, we present evidence that yeast epsin 2 (Ent2) plays a signaling role during cell division. We observed that overexpression of the ENTH domain of Ent2 (ENTH2), but not Ent1, promoted the formation of chains of cells and aberrant septa. This dominant-negative effect resulted from ENTH2-mediated interference with septin assembly pathways. We mapped the ENTH2 determinants responsible for induction of the phenotype and found them to be important for efficient binding to the septin regulatory protein, Bem3. Supporting a physiological role for epsin 2 in cell division, the protein localized to sites of polarized growth and cytokinesis and rescued a defect in cell division induced by Bem3 misregulation. Collectively, our findings provide a potential molecular mechanism linking endocytosis (via epsin 2) with signaling pathways regulating cell division.

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Section: Overexpression Of Enth2 In Yeast Cells Induces Defects In Cementioning

confidence: 99%

The yeast endocytic protein Epsin 2 functions in a cell-division signaling pathway

Mukherjee

Coon

Edwards

et al. 2009

Journal of Cell Science

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“…Yeast were grown in minimal medium (0.67% yeast nitrogen base without amino acids, Difco) supplemented with the appropriate amino acids and 2% galactose. For experiments designed to measure the ribozyme concentration dependence of intracellular cleavage rates, galactose was combined with different concentrations of sucrose or glucose to achieve different levels of chimeric ribozyme snoRNA expression (Ronicke et al 1997).…”

Section: Plasmid Construction and Propagationmentioning

confidence: 99%

Kinetic analysis of ribozyme–substrate complex formation in yeast

Yadava¹,

Mahen²,

Fedor³

2004

RNA

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Many RNA-mediated reactions in transcription, translation, RNA processing, and transport require assembly of RNA complexes, yet assembly pathways remain poorly understood. Assembly mechanisms can be difficult to assess in a biological context because many components interact in complex pathways and individual steps are difficult to isolate experimentally. Our previous studies of self-cleaving hairpin ribozymes showed that kinetic and equilibrium parameters measured in yeast agree well with parameters measured in vitro under ionic conditions that mimic the intracellular environment. We now report studies of intermolecular reactions with ribozyme and target sequences expressed in yeast as separate chimeric U3 snoRNAs. In this system, intracellular cleavage rates reflect the kinetics of ribozyme-substrate complex formation through annealing of basepaired helices. Second-order rate constants increased with increasing helix length for in vitro reactions with 2 mM MgCl 2 and 150 mM NaCl and in vivo but not in reactions with 10 mM MgCl 2 . Thus, efficient RNA complex formation required a larger extent of complementarity in vivo than in vitro under conditions with high concentrations of divalent cations. The most efficient intracellular cleavage reactions exhibited second-order rate constants that were 15-to 30-fold below rate constants for cleavage of oligonucleotides in vitro. Careful analysis of structural features that influence cleavage efficiency points to substrate binding as the rate-determining step in the intracellular cleavage pathway. Second-order rate constants for intermolecular cleavage agree well with diffusion coefficients reported for U3 snoRNPs in vivo suggesting that complex formation between chimeric ribozyme and substrate snoRNPs in yeast nuclei is diffusion limited.

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“…The complete coding sequences of AtSH3P1, AtSH3P2, and AtSH3P3 were cloned into p413MET25 under the control of the MET25 promoter (Ronicke et al, 1997) and transformed into yeast strain BY508 (with a deletion of the RVS167 gene). Both BY508 and BY263 (wild type) also were transformed with the empty p413MET25 as control.…”

Section: Yeast Complementationmentioning

confidence: 99%

Role of SH3 Domain-Containing Proteins in Clathrin-Mediated Vesicle Trafficking in Arabidopsis

Lam¹

2001

The Plant Cell Online

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Use of conditional promoters for expression of heterologous proteins in Saccharomyces cerevisiae

Cited by 34 publications

References 13 publications

The yeast endocytic protein Epsin 2 functions in a cell-division signaling pathway

The yeast endocytic protein Epsin 2 functions in a cell-division signaling pathway

Kinetic analysis of ribozyme–substrate complex formation in yeast

Role of SH3 Domain-Containing Proteins in Clathrin-Mediated Vesicle Trafficking in Arabidopsis

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