Team:Valencia Biocampus/Yeast

THE IDEA

Our aim in this part of the project is to detect when the yeast starts to ferment. At the end of the project we will be capable of “asking” the yeast if there is still any glucose in the media or not by the addition of H2O2. Furthermore we will be able to know how long the media has ran out of glucose. In conclution, this project allows us to know how much time has elipsed since the fermentation began.

To do this, we are going to use two gene constructions:

The ADH2 promoter fused to the YAP1 protein coding sequence. The protein YAP1 is a yeast transcription factor regulator of H2O2 adaptative response. It is stored in the citoplasm in normal conditions and, in presence of H2O2, is transported to the nucleous actting as a transcription factor. The ADH2 promoter is activated in abscence of glucose.

So, complete disappearence of glucose [] the production of YAP1 in the citoplasm whose concentration increases if the lack of glucose continues (we work with delta-yap1 strain).

The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence. The green fluorescent protein can be detected by fluorencent emission. The tiorredoxin reducase promoter is activated by two transcriptional factors (YAP1 and SKN7 in the oxidative form), both only bind to the promoter if H2O2 is previously added to the media.

MOLECULAR MECHANISMS

Fermentative response

This construction has three parts: (1) the first transcription factor binding site inside the promoter (UAS1), (2) the second transcription factor binding site (UAS2/CSRE), (3) the coding sequence which contains the yeast AP-1 protein (YAP1). We use the promoter of the ADH2 regulated by two trans-acting elements, both necessary for maximal promoter expression in absence of a fermentable carbon source [1].

When is the protein synthesized? Our construction is repressed several hundred-fold in the presence of glucose so, the transcription of YAP1 protein is initiated once the glucose in the medium is exhausted.

The molecular mechanism underlying this phenomenon is as follows: There are two transcriptional factors which regulates the promoter: ADR1 a carbon source-responsibe zinc-finger [2] and CAT8 [3]. In the absence of glucose both cis-acting sites in the promoter are bound cooperatively by the transcriptional activators. ADR1 binds to the UAS1 site while CAT8 binds to the UAS2/CSRE site and regulates positively the transcription of YAP1 protein by the activation of the ADH2 promoter [4]. The presence of glucose downregulates the levels of the transcription factors (ADR1 and CAT8) which results in the depletion of the production of YAP1 protein by the repression of the ADH2 promoter.

1. Tachibana C, et al. (2005) Combined global localization analysis and transcriptome data identify genes that are directly coregulated by Adr1 and Cat8.Mol Cell Biol 25(6):2138-46
2. Denis CL and Young ET (1983) Isolation and characterization of the positive regulatory gene ADR1 from Saccharomyces cerevisiae. Mol Cell Biol3(3):360-70.
3. Hedges D, et al. (1995) CAT8, a new zinc cluster-encoding gene necessary for derepression of gluconeogenic enzymes in the yeast Saccharomyces cerevisiae. Mol Cell Biol 15(4):1915-22
4. Walther K and Schuller HJ (2001) Adr1 and Cat8 synergistically activate the glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast Saccharomyces cerevisiae. Microbiology 147(Pt 8):2037-44

When is the protein synthesized? We obtain the green fluorescent protein by the addition of hydrogen peroxide (H₂O₂).

The molecular mechanism underlying this phenomenon is as follows: YAP1 transcription factor is regulated by subcellular localization. Under normal conditions an exporting receptor (Crm1) escorts YAP1 from the nucleus to the cytoplasm where it is stored. In the presence of hydrogen peroxide, another protein (Hyr1) catalyzes the formation of an intramolecular disulfide bond in YAP1 [2]. This conformational change allows YAP1 to accumulate in the nucleus and binds to the TRR promoter by a bZIP DNA-binding domain. In the other side, Skn7 transcriptional factor is constitutively nuclear. In the presence of hydrogen peroxide it is able to associate with the TRR promoter. Some studies suggest that the formation of the kn7+ yap1+ DNA complex requires more than both transcription factor binding to the promoter in close proximity, and the fact that both proteins interact directly. In conclusion, the two transcriptional factors require only one biding site in the promoter [3]. Once the two proteins are bound to the TRR promoter the transcription of the green fluorescent protein starts.

1. Jaekwon Lee, Christian Godon, Gilles Lagniel, Daniel Spector, Jérome Garin, Jean Labarre and B. Toledano (1999) Yap1 and Skn7 control two specialized oxidative stress response regulons in yeast. The journal of biological chemistry 23(274): 16040-16046.
2. Kuge S, et al. (1997) Regulation of yAP-1 nuclear localization in response to oxidative stress. EMBO J 16(7):1710-20
3. K. E. Mulford and J. S. Fassler (2011) Association of the Skn7 and Yap1 transcriptiom factors in the Saccharomyces cerevisiae oxidative Stress response. Eukaryotic cell 10(6):761

OUTLINE

1. We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein which comes in the bacterium plasmid pUC57.
2. We already had the Yeplac181 and Yep352 yeast vectors in our laboratory.
3. We carried out four transformations in E. coli, one for each DNA molecules (the two constructions and the two vectors), in order to clone them.See the Transformation Protocol Using Heat Shock
4. We obtained several E. coli colonies in four dishes and took some colonies of each DNA (two constructions and two vectors) to grow them in liquid medium overnight at 37 ºC in a shake chamber.
5. The next day we extracted the DNA molecules. See the Mini-prep Protocol.
6. We obtained the purified constructions (both in pUC57 plasmid) and the purified yeast vector (YEplac181 and YEp352).
7. We digested the four DNA molecules with restriction enzymes EcoRI and PstI. See the digestion protocol.
8. We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the ligation Protocol.
9. The day after, we transformed E.Coli with the ligation in order to amplify and store the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181)
10. We took some of the colonies to grow them in liquid medium overnight at 37ºC in a shake chamber
11. The next day, we extracted the DNA. See the Mini-prep Protocol.
12. After the final purified constructions were obtained, we checked it by electrophoresis and sequenced them.
13. We introduced one of the DNA constructions in yeast. See the Yeast transformation protocol.
14. We checked the presence of the construction by PCR. See the protocol here.
15. We used the obtained yeast in that moment and transformed it with the second construction. See the Yeast transformation protocol.
16. After that, we used a PCR protocol to check the presence of both constructions. In that moment, we transferred some colonies of these yeast to grow them in YPD
17. We measured the fluorescence at different glucose concentrations.
18. We obtained a curve relating these values.