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Review
. 2018 Feb 19;7(2):14.
doi: 10.3390/cells7020014.

The Yeast Saccharomyces cerevisiae as a Model for Understanding RAS Proteins and their Role in Human Tumorigenesis

Giulia Cazzanelli  1 , Flávia Pereira  2   3   4 , Sara Alves  5   6   7 , Rita Francisco  8 , Luísa Azevedo  9   10   11   12 , Patrícia Dias Carvalho  13   14   15 , Ana Almeida  16 , Manuela Côrte-Real  17 , Maria José Oliveira  18   19 , Cândida Lucas  20 , Maria João Sousa  21 , Ana Preto  22
Affiliations
Review

The Yeast Saccharomyces cerevisiae as a Model for Understanding RAS Proteins and their Role in Human Tumorigenesis

Giulia Cazzanelli et al. Cells. .

Abstract

The exploitation of the yeast Saccharomyces cerevisiae as a biological model for the investigation of complex molecular processes conserved in multicellular organisms, such as humans, has allowed fundamental biological discoveries. When comparing yeast and human proteins, it is clear that both amino acid sequences and protein functions are often very well conserved. One example of the high degree of conservation between human and yeast proteins is highlighted by the members of the RAS family. Indeed, the study of the signaling pathways regulated by RAS in yeast cells led to the discovery of properties that were often found interchangeable with RAS proto-oncogenes in human pathways, and vice versa. In this work, we performed an updated critical literature review on human and yeast RAS pathways, specifically highlighting the similarities and differences between them. Moreover, we emphasized the contribution of studying yeast RAS pathways for the understanding of human RAS and how this model organism can contribute to unveil the roles of RAS oncoproteins in the regulation of mechanisms important in the tumorigenic process, like autophagy.

Keywords: KRAS; RAS proteins; S. cerevisiae; autophagy; colorectal cancer; homologues; model.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The study of human proteins putatively involved in disease using S. cerevisiae as model can be summarized in three main methodologies. If the human protein has a yeast counterpart, the yeast protein can be studied in its environment and its function can be compared with the one in human cells, or the human gene can be cloned and expressed in yeast, in order to be studied in a neutral environment. Also human proteins that do not have a yeast orthologue can be cloned in yeast, especially with the purpose of finding their inhibitors or activators. In every case, the discoveries made in yeast need further validation in human cells.
Figure 2
Figure 2
Yeast and human RAS proteins present a highly conserved G domain, which is responsible for GTP/GDP binding. Human RAS proteins are also highly similar among them, HRAS showing 91% of amino acid identity in the G-domain to NRAS and 93% to KRAS. Yeast Ras proteins are bigger, having more than 100 extra amino acids, but still present around 60% amino acid identity in the G-domain to human RAS proteins.
Figure 3
Figure 3
Comparison of RAS proteins between human (Hs) and yeast (Sc) species. (a) Alignment of human KRAS, NRAS and HRAS with yeast Ras1 and Ras2. Identical residues at homologous positions are shown in blue. The Gly12 and Gly13 sites are highlighted in red. The CAAX box is highlighted in green. The lysine repeats of KRAS4A are highlighted in pink. Human sequences were obtained from Ensembl (KRAS4B: ENSP00000308495, KRAS4A: ENSP00000256078, NRAS: ENSP00000358548 and HRAS: ENSP00000309845) and yeast sequences from the Saccharomyces Genome Database (RAS1: YOR101W and RAS2: YNL098C). Sequences were aligned in Geneious 5.5.8 [94], using Muscle [95]. (b) Models of S. cerevisiae Ras1 (blue) and Ras2 (purple) using human KRAS structure (3GFT) as a template (green). (c) The three structures are shown superimposed, revealing the fold similarity. GDP and Mg sites are shown in orange.
Figure 3
Figure 3
Comparison of RAS proteins between human (Hs) and yeast (Sc) species. (a) Alignment of human KRAS, NRAS and HRAS with yeast Ras1 and Ras2. Identical residues at homologous positions are shown in blue. The Gly12 and Gly13 sites are highlighted in red. The CAAX box is highlighted in green. The lysine repeats of KRAS4A are highlighted in pink. Human sequences were obtained from Ensembl (KRAS4B: ENSP00000308495, KRAS4A: ENSP00000256078, NRAS: ENSP00000358548 and HRAS: ENSP00000309845) and yeast sequences from the Saccharomyces Genome Database (RAS1: YOR101W and RAS2: YNL098C). Sequences were aligned in Geneious 5.5.8 [94], using Muscle [95]. (b) Models of S. cerevisiae Ras1 (blue) and Ras2 (purple) using human KRAS structure (3GFT) as a template (green). (c) The three structures are shown superimposed, revealing the fold similarity. GDP and Mg sites are shown in orange.
Figure 4
Figure 4
Inactive RAS proteins bound to GDP are localized in the cytoplasm. GEFs catalyze the liberation of GDP from the binding site, allowing GTP, more abundant in the cell, to bind instead. GTP-bound RAS are translocated to the membrane and activated. GAPs enhance the endogenous GTPase activity, hydrolyzing GTP to GDP. RAS is inactive again and goes back to the cytoplasm, where the cycle can begin again, upon proper stimulus.
Figure 5
Figure 5
All RAS isoforms undergo a first farnesylation, followed by single palmitoylation in the case of KRAS4A and NRAS, double palmitoylation in the case of HRAS, and no palmitoylation in the case of KRAS4B. This splicing variant is retained in the inner leaflet of the plasma membrane by electrostatic interaction between the positively charged lysines and the negatively charged phospholipids. The three palmitoylated isoforms first pass through the Golgi and are then transported to the membrane via vesicular trafficking. Yeast RAS proteins behave very similar to the palmitoylated human isoforms, also being palmitoylated.
Figure 6
Figure 6
Human RAS proteins are localized in different cellular compartments, almost always associated with membranes. KRAS and HRAS are found in the inner leaflet of the plasma membrane, associated with galectin-3 [151,152] and galectin-1 [153], respectively. Inactive HRAS is very often located in cholesterol-rich structures called caveolae. RAS proteins can also signal from the membranes of internal organelles, such as ER, Golgi, mitochondria and the endosomal system.
Figure 7
Figure 7
Activated human RAS proteins can activate multiple effectors, having different impact on cell fate. Among the most studied, activated RAF triggers pro-growth signaling through the MAP kinases cascade; PI3K leads to AKT activation and RALGDS stimulated cell cycle progression.
Figure 8
Figure 8
Ras1 and Ras2 are activated upon nutrient availability signaling, glucose in particular. The GEF proteins Cdc25 or Sdc25 catalyze the liberation of GDP and the binding of GTP. Active RAS proteins activate in turn adenylate cyclase, which produces cAMP. This second messenger binds to the inhibitory unit of PKA, releasing the catalytic unit, which phosphorylates multiple downstream targets, leading to the activation of a variety of cellular processes or to the inhibition of transcription factors that control stress response. The pathway can be inactivated by the hydrolysis of cAMP by the phosphodiesterases Pde1 and Pde2, and by the GTPase activity of the GAPs Ira1 and Ira2.
Figure 9
Figure 9
The activation of RAS proteins leads to G1 to S phase progression through the cell cycle in both human and yeast. In both cases, RAS stimulates the formation of functional complexes between cyclins and CDK.
Figure 10
Figure 10
Human RAS proteins protect the cells from apoptosis mainly through the activation of PI3K and the consequent activation of AKT. Among AKT multiple targets there are Rac, IKK, CREB and Bad. Rac can be activated also by Tiam 1, activating in turn NF-κB, an important pro-survival factor. AKT phosphorylates also Bad, promoting the inhibition of the pro-apoptotic factor 14-3-3 and the consequent inhibition of apoptosis, and CREB, promoting the transcription of pro-survival genes. CREB is phosphorylated also by Rsk, activated by RAF/MEK/ERK signaling cascade. In addition, MEK promotes the downregulation of the pro-apoptotic protein Par4, contributing to apoptosis inhibition.
Figure 11
Figure 11
Serial drop test of BY4741 strain expressing human KRAS. S. cerevisiae BY4741 wt, Δras1 and Δras2 were subjected to different stress conditions (stress stimulus described on the side of each box). The direction of the serial dilutions, from 10−1 to 10−5, proceeds from top to bottom. The image is representative of one of three independent experiments.
Figure 12
Figure 12
S. cerevisiae wt, Δras1 and Δras2 transformed with p416LGALS3 were grown on glucose for 28 h from O.D.600 0.05 up to stationary phase. The untransformed yeasts and the yeasts transformed with the empty plasmid (p416 Ø) were used as controls. Specific growth rates were estimated from log phase. Graphs show the average specific growth rate ± SD of three independent experiments. Statistical significant differences are shown: * (p-value ≤ 0.05) and ** (p-value ≤ 0.01).
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