High efficiency transformation of <i>Schizosaccharomyces pombe</i> pretreated with thiol compounds by electroporation

Suga, Minoru; Hatakeyama, Tatsuko

doi:10.1002/yea.753

Cited by 77 publications

(16 citation statements)

References 28 publications

(43 reference statements)

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“…For the HIS3 assays, we developed an electroporation protocol based on the methods of Suga and Hatakeyama 58,59. After electroporation, cells were plated on minimal selective medium (SD, Synthetic Dropout) lacking Leu and His with the appropriate amount of 3-AT to suppress background (Matchmaker™ One-Hybrid System).…”

Section: Methodsmentioning

confidence: 99%

Max-E47, a Designed Minimalist Protein That Targets the E-Box DNA Site in Vivo and in Vitro

Chen

Jong

et al. 2009

J. Am. Chem. Soc.

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Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nM Kd values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14 nM, 15 nM, 9 nM, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site.

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Section: Methodsmentioning

confidence: 99%

Max-E47, a Designed Minimalist Protein That Targets the E-Box DNA Site in Vivo and in Vitro

Chen

Jong

et al. 2009

J. Am. Chem. Soc.

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“…Transformation of S. pombe was performed as described previously (22). Cells were routinely grown at 30°C in complete (YPD medium or Edinburgh's minimal medium (EMM) supplemented appropriately (i.e.…”

Section: Methodsmentioning

confidence: 99%

A Common Highly Conserved Cadmium Detoxification Mechanism from Bacteria to Humans

Prévéral

Gayet

Moldes

et al. 2009

Journal of Biological Chemistry

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Cadmium poses a significant threat to human health due to its toxicity. In mammals and in bakers' yeast, cadmium is detoxified by ATP-binding cassette transporters after conjugation to glutathione. In fission yeast, phytochelatins constitute the cosubstrate with cadmium for the transporter SpHMT1. In plants, a detoxification mechanism similar to the one in fission yeast is supposed, but the molecular nature of the transporter is still lacking. To investigate further the relationship between SpHMT1 and its co-substrate, we overexpressed the transporter in a Schizosaccharomyces pombe strain deleted for the phytochelatin synthase gene and heterologously in Saccharomyces cerevisiae and in Escherichia coli. In all organisms, overexpression of SpHMT1 conferred a markedly enhanced tolerance to cadmium but not to Sb(III), AgNO 3 , As(III), As(V), CuSO 4 , or HgCl 2 . Abolishment of the catalytic activity by expression of SpHMT1 K623M mutant suppressed the cadmium tolerance phenotype independently of the presence of phytochelatins. Depletion of the glutathione pool inhibited the SpHMT1 activity but not that of AtHMA4, a P-type ATPase, indicating that GSH is necessary for the SpHMT1-mediated cadmium resistance. In E. coli, SpHMT1 was targeted to the periplasmic membrane and led to an increased amount of cadmium in the periplasm. These results demonstrate that SpHMT1 confers cadmium tolerance in the absence of phytochelatins but depending on the presence of GSH and ATP. Our results challenge the dogma of the two separate cadmium detoxification pathways and demonstrate that a common highly conserved mechanism has been selected during the evolution from bacteria to humans.Cadmium is a trace element, the presence of which in the environment is essentially due to human activities. It is a highly toxic non-biological heavy metal able to enter living cells via transporters usually used for the uptake of essential cations such as calcium, iron, zinc, and so forth (1). The reactivity of cadmium with thiol groups and its ability to displace essential biological metals result in oxidative stress and eventually cell death (2). To cope with cadmium toxicity, living organisms have developed different strategies.In animals, as in the bakers' yeast cytoplasmic cadmium is complexed with the thiol tripeptide glutathione, a general redox regulator (3, 4). Bis(glutathionato)-cadmium complexes (Cd-GS 2 ) 4 are then driven from the cytoplasm to lesser sensitive cellular compartments by dedicated transporters. The prototypical transporter of Cd-GS 2 is the GS-X pump, ScYCF1, in Saccharomyces cerevisiae (5) and, even if still controversial, to a lesser extent HsMRP1 in humans (6). HsMRP1 probably acts as an efflux pump at the plasma membrane, delivering cadmium in the extracellular medium, whereas ScYCF1 allows sequestration of cadmium into the vacuole (5). A study of a deficient Scycf1 strain has shown that it was extremely cadmium-sensitive, pointing to a major role of ScYCF1 in cadmium tolerance and detoxification (5). Additionally, ScYCF1 wa...

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“…Electrocompetent cells of the reporter strain were prepared following a protocol based on the methods described by Suga and Hatakeyama [58], [59]. Yeast cells were grown overnight in YPDA liquid medium (20 g/L Difco peptone, 10 g/L yeast extract, 0.009% adenine hemisulphate).…”

Section: Methodsmentioning

confidence: 99%

Hybrids of the bHLH and bZIP Protein Motifs Display Different DNA-Binding Activities In Vivo vs. In Vitro

Chow

Shahravan

et al. 2008

PLoS ONE

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Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim) protein Arnt fused to the leucine zipper (LZ) dimerization domain from bZIP (basic region-leucine zipper) protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper) proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H), transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed), as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K d 148.9 nM and 40.2 nM, respectively), but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly α-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60–70 aa). Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

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High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation

Cited by 77 publications

References 28 publications

Max-E47, a Designed Minimalist Protein That Targets the E-Box DNA Site in Vivo and in Vitro

Max-E47, a Designed Minimalist Protein That Targets the E-Box DNA Site in Vivo and in Vitro

A Common Highly Conserved Cadmium Detoxification Mechanism from Bacteria to Humans

Hybrids of the bHLH and bZIP Protein Motifs Display Different DNA-Binding Activities In Vivo vs. In Vitro

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