Ras Signaling in Yeast

Abstract

Since the study of yeast RAS and adenylate cyclase in the early 1980s, yeasts including budding and fission yeasts contributed significantly to the study of Ras signaling. First, yeast studies provided insights into how Ras activates downstream signaling pathways. Second, yeast studies contributed to the identification and characterization of GAP and GEF proteins, key regulators of Ras. Finally, the study of yeast provided many important insights into the understanding of C-terminal processing and membrane association of Ras proteins.

Yeast RAS Proteins

The Saccharomyces cerevisiae genome contains 2 RAS genes, RAS1 and RAS2.^1,2 They encode proteins of 309 and 322 amino acid residues, respectively (Fig. 1). N-terminal portions of these proteins have significant homology to the mammalian Ras. This region contains G1 to G5 boxes, short stretches of amino acids that are involved in the recognition of guanine nucleotide and phosphate. Guanine nucleotide binding and GTPase activities of Ras1 and Ras2 proteins have been characterized.³ In the C-terminal third of the molecule, yeast Ras proteins diverge significantly with the mammalian Ras. The sequence very close to the C-terminus including the terminal 4 amino acids that constitute the CAAX motif (C is cysteine, A is aliphatic amino acid, and X is the C-terminal amino acid) is important for posttranslational modifications that facilitate their membrane association. Ras1 and Ras2 have similar functions, but their expressions differ. Expression of Ras1 is shut down when cells are grown in media containing nonfermentable carbon sources such as glycerol or pyruvate.⁴ Mammalian ras can suppress the loss of yeast RAS, supporting the functional similarity between the yeast and mammalian genes.^5,6 On the other hand, the yeast RAS gene product containing an amino acid change at the residue corresponding to glycine-12 of mammalian Ras can transform NIH3T3 cells provided that the extra sequence in the C-terminal region of yeast Ras is deleted.⁵

Figure 1.

Structure of Ras proteins. Saccharomyces cerevisiae encodes 2 Ras proteins, Ras1 and Ras2. Schizosaccharomyces pombe encodes only one Ras protein called Ras1. Human H-Ras is shown to indicate that the S. pombe Ras1 resembles the human Ras more. C-terminal sequences are shown including the 4 C-terminal amino acids that constitute the CAAX motif. Cysteines adjacent to the CAAX motif are palmitoylated.

Schizosaccharomyces pombe has only one ras gene called ras1.^7,8 The encoded protein has 219 amino acid residues; thus, its size is close to that of mammalian Ras protein (Fig. 1). A human ras cDNA sequence can complement a ras1 ⁻ strain, demonstrating functional similarity between the fission yeast and human genes.

RAS Downstream Effectors and Signaling Pathways

Saccharomyces cerevisiae

The RAS genes are essential for growth; thus, ras1 ⁻ ras2 ⁻ double mutants are nonviable.^9,10 The ras2 ⁻ mutants do not grow on a nonfermentable carbon source, as they will be defective in both Ras1 and Ras2 in nonfermentable carbon sources. Cells with a temperature-sensitive RAS2 mutation as well as ras1 ⁻ mutation are blocked in the G1 phase of the cell cycle and accumulate as unbudded cells at nonpermissive temperatures. Mutations of the RAS2 gene cause accumulation of storage carbohydrates and sporulation even on rich media. On the other hand, yeast cells expressing an activating mutant of Ras2, Ras2^val19, exhibit 1) reduced sporulation as detected by a decreased glycogen storage level, 2) sensitivity to heat shock, and 3) sensitivity to nutrient starvation.⁹ The amount of cAMP inside the cell is decreased in the ras mutants and increased in the mutant expressing Ras2^val19.⁹

Both Ras1 and Ras2 function to activate adenylate cyclase, which is associated with a protein called CAP^10,11 (Fig. 2A). Adenylate cyclase synthesizes cAMP from ATP, which then binds Bcy1 protein, a regulatory subunit of protein kinase A, to activate PKA. Three genes, TPK1, TPK2, and TPK3, encode the catalytic subunit of protein kinase A. Phosphorylation of substrates leads to the regulation of a variety of functions including cell cycle progression. cAMP is hydrolyzed by a low-affinity phosphodiesterase encoded by the PDE1 gene as well as by a high-affinity phosphodiesterase encoded by the PDE2 gene.^12,13 The production of cAMP is also regulated by the Gα protein called Gpa2 that is activated by glucose.¹⁴

Figure 2.

Ras signaling pathways in Saccharomyces cerevisiae (A) and in Schizosaccharomyces pombe (B).

Adenylate cyclase is encoded by the CYR1 gene. This gene encodes a large membrane-bound protein of 2,026 amino acids. Four domains of the protein have been identified: the N-terminal, middle repetitive, catalytic, and C-terminal domains. The middle repetitive domain consists of a repeat of a 23-residue amphipathic leucine-rich motif called the LRR domain (amino acids 674-1,300). This LRR domain is the primary site of interaction with Ras. The N-terminal 81 amino acids of the LRR domain (676-756) have been identified as a Ras-associating domain (RAD), a motif of about 100 amino acids conserved among Ras-binding regions of mammalian RalGDS, Rin1 and afadin/AF-6, by a computer algorithm–based search.¹⁵

The RAS/cAMP/PKA pathway regulates a variety of processes including cell cycle progression and life span.^9,10,16 Recent studies identified a number of other cellular processes regulated by the RAS/cAMP/PKA pathway. For example, this signaling pathway is reported to regulate the polarity of actin cytoskeleton through the stress response pathway.¹⁷ It also controls spore morphogenesis.¹⁸ Regulation of the activity of amino acid transporter Gap1 permease is reported.¹⁹ More recently, it has been shown that deregulated Ras signaling compromises DNA damage checkpoint recovery.²⁰

Phosphorylation of Ras proteins has been reported.²¹ This was initially shown by the detection of Ras proteins with decreased mobility on a SDS polyacrylamide gel. Labeling with radioactive phosphate showed that serine is phosphorylated. Further studies by Xiaojia and Jian²² showed that serine 214 of Ras2p is phosphorylated. A substitution of serine 214 to alanine resulted in enhanced sensitivity to heat shock, reduced glycogen, and increased cAMP, suggesting that phosphorylation is involved in the feedback regulation of the Ras-cAMP pathway.

Schizosaccharomyces pombe

The ras1 mutant cells are abnormally round and sterile. This is because of the involvement of Ras1 in 2 different downstream signaling pathways (Fig. 2B). Ras1 activates Byr2 protein, a MAP kinase kinase (MEKK). Byr2 activates another kinase called Byr1, which then activates the kinase Spk1, resulting in the regulation of pheromone signaling.²³ Thus, Ras1 activates the MAPKinase module, and this aspect is similar to the activation of the Raf/Mek/Erk by mammalian Ras. Byr2 is activated by Ste4 and Gpa1 in addition to Ras1. The solution structure of the Ras-binding domain of Byr2 has been determined by NMR.²⁴ The structure consists of 3 α-helices and a mixed 5-stranded β-pleated sheet. The first 7 canonic secondary structure elements form a ubiquitin superfold.²⁴

Ras1 also regulates Scd1/Ral1, a guanine nucleotide exchange factor (GEF) for Cdc42, a member of the Rho family GTPase^25,26 that activates the Shk1/Orb2 protein kinase.²⁷ The Ras1-Scd1 pathway is regulated by a scaffold protein, Scd2/Ral3.²⁸ One of the functions of Scd1 is to regulate spindle formation, and this is mediated by the interaction of Scd1 with Moe1.²⁹ Scd1 also interacts with Klp5 and Klp6 kinesins to affect cytokinesis.³⁰

Regulators of RAS Proteins: GAP and GEF

Saccharomyces cerevisiae

Yeast Ras1 and Ras2 are downregulated by Ira proteins (inhibitors of Ras).³¹ Two genes, IRA1 and IRA2, are present, and these gene products are very similar. A region of approximately 360 amino acids in the middle of these gene products, called the GAP domain, is responsible for activating intrinsic GTPase activity of Ras.³² Ira1 and Ira2 have similar functions; mutations of IRA1 and IRA2 have similar phenotypes that are consistent with the activation of RAS signaling, and the phenotypes of the double mutant have even more pronounced phenotypes. Importantly, mutations in IRA can suppress mutations in CDC25. Thus, double mutants consisting of ira ⁻ and cdc25 ⁻ are viable.

The study in yeast contributed significantly to the research on the regulator of Ras. The case in point can be seen with the NF1 gene. Mutations of this gene result in a genetic disorder called neurofibromatosis type I that is characterized by the uncontrolled growth of Schwann cells that wrap neurons. Symptoms of NF1 include occurrence of benign tumors called neurofibromas, café au lait spots, and bone disorders. In addition, learning disorders accompany the disease. When the NF1 gene was cloned, weak but significant similarity to the IRA genes of yeast S. cerevisiae was noted. NF1 protein contains a stretch of about 360 amino acid residues in the middle of the protein (GRD). This region is responsible for the GAP activity to downregulate the Ras protein converting GTP-bound Ras to GDP-bound Ras.³³ The GRD of NF1 can complement the mutations in the IRA genes. Expression of NF1-GRD suppresses heat shock sensitivity of the ira ⁻ mutants. Recombinant NF1-GRD exhibits the GAP activity towards Ras.

How Ira proteins are regulated is of interest. It has been reported that the Kelch proteins Gpb1 and Gpb2 bind to a conserved C-terminal domain of Ira1 or Ira2 and that this contributes to stabilizing Ira proteins.³⁴ Interestingly, Gpb is a Gβ mimic that forms a protein complex with Gpa2, a Gα subunit that is activated by a G protein–coupled receptor, Gpr1. Direct regulation of PKA by Gpb proteins is also reported.³⁵ A recent proteomics approach identified Gpb1 as a binding partner of Ira2, and this study showed that Gpb1 negatively regulates Ira2 by promoting its ubiquitin-dependent proteolysis.³⁶ Gpb2, on the other hand, positively regulates Ira2. Phan et al. extended this study to mammalian cells and showed that the ETEA/UBXD8 protein directly interacts with the NF1 gene product (neurofibromin) and negatively regulates it. Another interesting observation concerns the interaction of Ira1with Cyr1, an interaction that may be important for the membrane localization of Ira.³⁷

The CDC25 gene encodes an activator of Ras1 and Ras2 that acts as a GEF (GDP/GTP exchange factor) that promotes the exchange of bound GDP with GTP.^38,39 CDC25 is an essential gene that is required for cAMP production. Activating mutants of Ras2 such as Ras2^val19 can suppress mutations in CDC25, as the active Ras does not need an exchange factor to be activated. Another gene called SDC25 was shown to contain a RAS-activating (GEF) domain. However, this gene is dispensable, and the C-terminal portion of the gene product is responsible for the suppression.⁴⁰

Schizosaccharomyces pombe

The study in S. pombe supported the idea that Ras signaling is compartmentalized.⁴¹ As described before, Ras1 activates 2 different downstream signaling pathways, the Byr2-MAP kinase cascade and the Scd1-Cdc42 pathway. Ras1 mutants that are plasma membrane restricted activate the former pathway, while endomembrane-restricted Ras1 activates the latter pathway.⁴¹ Two different GEFs for Ras1, Ste6 and Efc25, have been identified.^42,43 Interestingly, these GEF proteins cause different downstream activation of Ras1.⁴⁴ Ste6 specifically activates Ras1 so that Byr2 is activated to influence mating (MAP kinase pathway). On the other hand, Efc25 activates the Ras1-Scd1 pathway to influence morphology (Cdc42 pathway). Thus, 2 different GEF proteins are used to specifically affect different downstream pathways in fission yeast.

C-terminal Processing and Membrane Association of RAS

Overall Processing Steps

Yeast Ras proteins are processed at their C-termini, and this is important for their membrane association and function. An overall process of this posttranslational modification is shown in Figure 3. Ras proteins are synthesized in the cytoplasm. The first event is the removal of methionine at the N-terminus, which presumably occurs cotranslationally. Then, a series of C-terminal modification events occur that includes farnesylation, C-terminal 3 amino acids removal, carboxyl methylation, and palmitic acid addition. This process is similar to what happens with the mammalian Ras. Thus, the study of yeast contributed significantly to the elucidation of C-terminal modification events for mammalian Ras.

Figure 3.

C-terminal processing of yeast Ras proteins. The figure also shows Saccharomyces cerevisiae genes (italicized) involved in each processing step.

Protein Prenylation

Ras proteins end with the C-terminal CAAX motif (C is cysteine, A is an aliphatic amino acid such as leucine or isoleucine, and X is the C-terminal amino acid), and farnesylation occurs on the cysteine in the CAAX motif. Farnesylation of Ras1 and Ras2 was shown by the labeling with mevalonic acid and its removal by rainy nickel followed by HPLC identification.⁴⁵ Farnesylation is characterized by protein farnesyltransferase, a heterodimeric enzyme consisting of α and β subunits that are encoded by the RAM2 and the DPR1/RAM1 genes, respectively.⁴⁵ The RAM2 gene is essential, as this gene product functions also as the α subunit of protein geranylgeranyltransferase type I.^46,47 The DPR1/RAM1 gene is not essential, but the mutants exhibit temperature-sensitive growth and sterility because of deficiency in the a-factor that signals mating response. Ras proteins largely remain cytoplasmic in the dpr1/ram1 mutants. Protein farnesyltransferase is responsible for the farnesylation of other proteins such as the γ subunit of heterotrimeric G proteins Ste18⁴⁸ and Rheb G protein.⁴⁹

Fission yeast Ras1 is farnesylated, and this is catalyzed by protein farnesyltransferase that is encoded by the cpp1 (β subunit) and the cwp1 genes (α subunit).⁵⁰ Mutations of these genes result in cytoplasmic accumulation of Ras1. Mutants defective in the cpp1 gene are temperature sensitive for growth, while mutants defective in the cwp1 gene are not viable. This is because the cwp1 gene product also functions as the α subunit of protein geranylgeranyltransferase type I.

Postprenylation Processes

Characterization of mature Ras2 demonstrated that the 3 C-terminal amino acids were removed and that the C-terminal residue was at the C-terminus.⁵¹ Furthermore, the C-terminal cysteine was methylated.⁵² Another study identified the occurrence of S-farnesylcysteine α-carboxylmethyl ester at the C-terminus of Ras2.⁵³ Genetic analysis of Ras and a-factor processing led to the identification of genes involved in these processes. The RCE1 gene encoding a protease called Ras-converting enzyme 1 that removes 3 C-terminal amino acids was identified.⁵⁴ Rce1 is an ER integral transmembrane protease. Then, the cysteine that has become the C-terminal residue is methylated by the STE14 gene product that encodes isoprenylcysteine carboxyl methyltransferase (Icmt), a 26-kDa integral membrane protein that contains 6 transmembrane-spanning segments.⁵⁵ Icmt is localized to the endoplasmic reticulum membrane. A dimerization motif is found in transmembrane 1 of Icmt, and it has recently been shown that Icmt forms a homodimer.⁵⁶

Fission yeast Icmt was identified from the studies of mutations that cause mating deficiency in h ⁻ cells but not in h ⁺ cells.⁵⁷ The mam4 gene encodes a protein of 236 amino acids with several potential membrane-spanning domains. The protein is 44% identical with Icmt encoded by the budding yeast STE14. This study also identified Xmam4, the gene for Icmt in Xenopus laevis.

Palmitoylation

Palmitoylation of S. cerevisiae Ras1 and Ras2 was demonstrated by carrying out palmitic acid labeling.⁵⁸ The incorporated radioactivity was shown to be palmitic acid by recovering the radioactivity by mild alkali treatment and by identifying the released palmitic acid using thin-layer chromatography. The enzyme that catalyzes this modification called protein palmitoyltransferase (PAT) was found to be a heterodimeric enzyme that is encoded by the ERF2 and ERF4 genes. Erf2p is a 41-kDa protein localized to the endoplasmic reticulum (ER) membrane, while Erf4 is a 26-kDa peripheral ER protein.⁵⁹ Erf2 and Erf4 form a complex, and this complex is responsible for carrying out palmitoylation of Ras2 using palmitoyl-CoA as a donor of a palmitoyl group. Human PAT that catalyzes palmitoylation of H-Ras and N-Ras proteins consists of the DHHC9 and GCP16 genes.⁶⁰ DHHC9 protein is an integral membrane protein possessing the DHHC-CRD domain, while GCP16 is a Golgi-localized membrane protein.⁶⁰

The Erf2 protein contains a conserved DHHC cysteine-rich domain (DHHC-CRD). Genes encoding proteins with the DHHC-CRD have been identified in genomic databases for worms, flies, and mammals. In mouse and human databases, about 20 genes have been identified to encode proteins with the DHHC-CRD motif.⁶¹ These proteins include Huntingtin interacting protein 14 (HIP14/DHHC17) and GODZ/DHHC3, a Golgi-specific DHHC zinc finger protein. Thus, the discovery of PAT genes in yeast led to the identification of a novel class of palmitoyltransferase enzymes that contain the DHHC motif.

Future Prospects

The study in yeast has contributed significantly to our understanding of the Ras signaling pathway in mammalian cells. The work on downstream effectors of Ras contributed to the idea of the Ras-binding domains and reinforced the idea that one of the functions of Ras is to activate the MAP kinase cascade. Fission yeast provided a model system for the idea of “compartmentalized signaling.” The hint that NF1 functions as a GAP for Ras came from the study of the Ira proteins. The study in yeast contributed to elucidating the C-terminal processing of Ras. Cloning of the gene encoding a subunit of protein farnesyltransferase was first carried out in yeast.⁶² Rce1,⁵⁴ Icmt,⁵⁵ and RasPAT⁵⁹ were discovered in yeast.

What future research in yeast could make an impact in the study of mammalian Ras? One area of current research in yeast concerns the effort to understand global signaling pathways. This systems biology approach will place Ras signaling in the context of global signaling events. This will lead to the understanding of signaling networks and how overall signaling will be affected by nutrients, energy, and stress conditions. Mathematical models of the cAMP pathway in S. cerevisiae have been proposed.⁶³ Further studies of this type should provide deeper understanding of the Ras signaling pathway.