Construction Of Reporter Gene In Alg6 Yeast Variants Biology Essay

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Chapter 6

6.1 Introduction

The unfolded protein response (UPR) regulates gene expression in response to stress in the endoplasmic reticulum (ER). Proteins entering the secretory pathway fold within the confines of the endoplasmic reticulum (ER). To support efficient folding, the ER maintains an environment enriched in chaperones, glycosylation enzymes, and oxidoreductases. Despite this optimized environment, an inevitable consequence of the large flux of proteins through the ER is that the folding process fails in about 30% of all newly synthesised proteins, resulting in the production of misfolded proteins. Two distinct processes have been described that help eukaryotic cells cope with this problem: The unfolded protein response (UPR) attempts to remediate misfolded molecules and failing that, ER-associated degradation (ERAD) disposes of irrevocably proteins by reverse translocation to the cytosolic 26S proteosome after tagging with ubiquitin (Figure 6.1) (Bonifacino and Weissman, 1998). It appears that several components of the translocon and cytosolic degradation machinery are shared by ERAD and other cellular processes.

The UPR remediation pathway activates the transmembrane kinase/nuclease Ire1p (Cox et al., 1993), causing the nonconventional splicing of HAC1 mRNA and the production of Hac1p, a bZIP transcription factor that induces the UPR target genes. This regulatory response of gene expression by the UPR allows the cell to tolerate folding stress by correcting unfolded proteins. While the mechanism by which the UPR signal is transmitted from the ER to the nucleus is well characterized, it is less clear how this response corrects misfolding. Of the UPR target genes identified so far (Travers et al., 2000) most encode ER-resident chaperones, as might be expected for a response to the accumulation of unfolded proteins (Chapman et al., 1998). In addition, components of the phospholipid biosynthetic pathways are targets, suggesting a role for the UPR in maintenance and biogenesis of the ER membrane. Identification of the complete set of UPR target genes and their interaction networks thus promises to provide insight into the means by which the cell copes with folding stress and adjusts the capacity of protein folding in the ER according to need.

Figure 6.1: The Unfolded Protein Response. The UPR acts to reduce level of misfolded protein by enhancing the protein to fold to the native state. It also promotes transit to the distal secretory pathway and enhancing the rate of ERAD while simultaneously reducing the formation of misfolded proteins.

The aim of this chapter was to observe the UPR stress caused by deletion of the yeast alg6 gene or C998T point mutation on the human ALG6 in yeast. By constructing an integrated reporter gene in which GFP was driven by four copies of the unfolded protein response element (UPRE) (Figure 6.2). A loss of ERAD function would result in an accumulation of unfolded proteins in the ER and chronic activation of the UPR. This chapter describes activation of the UPR in yeast cells lacking ALG6 gene or yeast cells with human CDG-Ic disorder using a sensitive reporter of UPR activation.

Figure 6.2: Construction of reporter gene in ALG6 yeast variants. The reporter construct consisted of the gene encoding GFP driven by four repeats of the UPRE.

6.2 Construction of reporter gene in ALG6 yeast variants

The four repeats of the unfolded protein response element linked to the GFP were linked to the URA3 gene for positive selection of yeast transformants on synthetic media lacking uracil. This fragment was inserted at the met15 knockout locus in the yeast genome (Figure 6.3). The RFP marker, mCherry was used to label the cytoplasm and this marker was under the control of the constitutive TEF2 promoter and also acts as a neutral marker to normalize GFP expression levels.

Figure 6.3: Integration of the 4xUPRE-GFP-URA3 at yeast met15Δ0 locus by homologous recombination. The 4 x UPRE linked to GFP and URA3 gene. The product with the flanking MET 15 UTR regions were integrated at the met15Δ0 locus in yeast by homologous recombination.

6.3 Screening

The alg6 variants with RFP reporter were replicated on fresh SD media plates and grown overnight for imaging purposes. Screening for GFP expression against neutral RFP expression was carried out using 384-well microscope clear bottom plates (Perkin Elmer Cellcarrier) with each well filled with 50 µL SC liquid media. Images of each alg6 variant were acquired using the OPERA automated confocal microscope in which GFP was excited at 488 nm and RFP was excited at 561 nm.

6.4 Results

Comparison of steady state levels of GFP fluorescence in the Δalg6 yeast strain to those of the wildtype yeast cells revealed increase in the activation of unfolded protein response under normal growth conditions, indicating the stress level of yeast cells with deletion of the ALG6 gene.

Figure 6.4: Microscopic analysis of wildtype and Δalg6 yeast strains expressing 4xUPRE-GFP with mCherry cytoplasmic RFP.

Representative fluorescent expression experiment in wildtype yeast cells (left). UPRE-GFP image (cytoplasm; top left), mCherry cytoplasmic RFP (cytoplasm; middle left), Fluorescence images of GFP (top left) and RFP (middle left) were taken and merged (bottom left).

Representative fluorescent expression experiment in Δalg6 yeast cells (right). UPRE-GFP image (cytoplasm; top right), mCherry cytoplasmic RFP (cytoplasm; middle right), Fluorescence images of GFP (top right) and RFP (middle right) were taken and merged (bottom right).

Replacing yeast ALG6 gene with the human ALG6 gene in yeast caused only low level activation of the unfolded protein response compared to the unfolded protein response seen in Δalg6. The level of GFP florescence in yeast strain with CDG-Ic mutation compared to those of the yeast cells with human ALG6 gene revealed an increase in the activation of unfolded protein response under normal growth conditions, indicating the stress level of yeast cells with C998T point mutation on the human ALG6 gene (Figure 6.5).

Figure 6.5: Microscope assay of yeast with human ALG6 strain and yeast with CDG-Ic mutation expressing 4xUPRE-GFP with mCherry cytoplasmic RFP.

Representative fluorescent expression experiment in yeast cells with human ALG6 gene (left). UPRE-GFP image (cytoplasm; top left), mCherry cytoplasmic RFP (cytoplasm; middle left), Fluorescence images of GFP (top left) and RFP (middle left) were taken and merged (bottom left).

Representative fluorescent expression experiment in yeast cells with CDG-Ic disorder (right). UPRE-GFP image (cytoplasm; top right), mCherry cytoplasmic RFP (cytoplasm; middle right), Fluorescence images of GFP (top right) and RFP (middle right) were taken and merged (bottom right).

A script was developed and used to measure the GFP and RFP intensity under unfolded protein response in yeast ALG6 variants (Appendices 11.5).

Figure 6.6: GFP and RFP intensity upon activation of the Unfolded Protein Response. The column bar graph in green represents GFP and column bar graph in red represents RFP. (* = p value < 0.05, ** = p value < 0.005, *** = p value < 0.0005). Horizontal bar represents comparison between two bars.

The levels of UPR GFP florescence in the Δalg6 yeast strain compared to those of the wildtype yeast cells revealed a 5.5 fold increase in the activation of unfolded protein response (p value = 0.0027) under normal growth conditions (Figure 6.6). Replacement of yeast ALG6 gene by human ALG6 gene did not cause such a distinct unfolded protein response, but still the GFP intensity difference was 2.1 fold (p value = 0.0112) when compared to wildtype yeast cells. C998T point mutation in the human ALG6 gene increased the activation of unfolded protein response by 5.3 fold (p value = 0.001) indicating the stress level of yeast cells with CDG-Ic disorder. Comparison between the RFP intensity between ALG6 variants and wildtype revealed around 2 fold increase. Hence normalization of GFP to the neutral RFP intensity was performed.

Figure 6.7: GFP/RFP intensity ratio between wildtype yeast, yeast with alg6 knockout, yeast with human ALG6 gene and yeast with CDG-Ic mutation upon activation of the Unfolded Protein Response. (* = p < 0.05, ** = p value < 0.005, *** = p value < 0.0005). Horizontal bar represents comparison between two bars.

Comparison of levels of GFP/RFP intensity ratio in the Δalg6 yeast strain to those of the wildtype yeast cells reveals 3.2 fold induction (p value = 0.0041) of the UPR under normal growth conditions (Figure 6.7). This substantial increase in the UPR indicated the important role of the glycosylation mediated by the -1, 3 glucosyltransferase enzyme that is altered in CDG-Ic, responsible for assisting in the folding of glycoproteins. The GFP/RFP intensity difference between wildtype yeast and yeast with human ALG6 gene was not significant (p value = 0.0539). This could be because the overall identity and similarity between the two orthologs (32% and 51%, respectively) provides sufficient folding capability. The C998T point mutation on the human ALG6 by comparison showed significant increase in UPR (p value = 0.0003). The -1, 3 glucosyltransferase is required for the addition of the first of three glucose residues to LLO, and without the first glucose, further glucosylation is prevented. The nonglucosylated precursor oligosaccharide is a poor substrate for the oligosaccharyltransferase complex and is inefficiently transferred to proteins. Furthermore, the absence of glucose on the oligosaccharides affects the quality control and folding of the proteins resulting into protein accumulation and degradation and significant activation of the UPR.

6.5 Conclusion

In the ER, proteins fold, oligomerise and pass on to the later secretory pathway. Persistently misfolded proteins are eliminated by the ERAD machinery. Cellular stress results in accumulation of misfolded proteins and activates the UPR. Our observation revealed 3.2 fold induction of the UPR as a result of deletion of the ALG6 gene in S.cerevisiae whereas the human ALG6 gene in yeast was largely able to substitute for the yeast gene. Yeast cells with the CDG-Ic mutation revealed a 2.5 fold induction of the UPR. The UPR acts to reduce level of misfolded protein by enhancing protein to fold to the native state and promoting transit to the distal secretory pathway. UPR also enhances the rate of ERAD while simultaneously reducing the formation of misfolded proteins.

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