Vid30 complex participation in the degradation of CDC25 in yeast and their role in Ras/ cAMP/ PKA pathway
Background:
Unicellular organisms such as Saccharomyces cerevisiae in its total life cycle is exposed to diverse and changing nutrient conditions. Thus, in order to survive and achieve a competitive advantage, it has adapted itself such that it not only uses the wide range of available nutrient sources but also the sources that are nutrient rich. It mainly uses fructose and glucose by rapidly fermenting to alcohol. In response to these nutrient conditions, the intracellular trafficking transcription and turnover of protein from nutrient transporters (e.g. Hxts) get regulated. Therefore, when the availability of the glucose increases, the expression of low- affinity hexose transporter (Hxt3p) also increases (Snowdon, 2012). This then localizes in the plasma membrane (i.e., either the external nutrient substrate binds itself to the sensor of the plasma membrane or the internal nutrient intermediate binds itself to a cytosolic sensor), where it helps in transportation of sugars (Snowdon, 2008). However, when the shift occurs, it consumes the sole carbon source, ethanol, maltose or galactose, following which the Hxt3p undergoes endocytosis and is transported to the vacuole where degradation takes place, thereby repressing its expression (Snowdon, 2012).
Contrastingly, the expression of high- affinity hexose transporter Hxt7 increases and starts functioning in the plasma membrane where the nitrogen availability is abundant and the glucose is limited. In such scenario, it utilizes glutamine or ammonia. The transcription of Hxt7 can be decreased by creating a nitrogen starvation environment or by rapamycin treatment, thereby targeting protein degradation. Thus, in order to survive in both nutrient- rich and nutrient- starved conditions, Saccharomyces cerevisiae have adapted itself with nutrient- signaling and nutrient- sensing mechanisms, which can activate or inactivate related genes and thereby the protein production (Boles, 1993). However, the exact process is still unknown. Studies have reported that in order to adapt well to the different nutrient environments, Saccharomyces cerevisiae uses different signaling pathways such as the Ras/cAMP/Protein kinase A (PKA) and TORC1 pathways. The Gid and Vid proteins comprise of the vid30 complex, which is an E3 ubiquitin ligase help in the nutrient- dependent degradation of the FBPase and Mdh2p (gluconeogenic enzymes) when the glucose- starved cells are provided with abundant glucose (Krampe, 2002; Hung, 2004). The Vid30c components are highly responsible for the endocytosis and Hxt3 and Hxt7 degradation in response to the various nutrient signals. In this review, the role of Vid30 complex in the CDC25 degradation in yeast will be discussed. The review will also discuss their role in Ras/ cAMP/ PKA pathway.
Glucose acts as the key carbon source for different cells that provides them with energy and helps in building necessary cellular components. It also helps in cell regulation. The glycolysis and the gluconeogenesis pathways are the major pathways used by different organisms to metabolize carbohydrates. While the glucose helps in cell regulation, the cyclic adenosine monophosphate (cAMP) and Ras signaling helps in cell- growth coordination and proliferation by nutrient sensing. The activity of the Ras proteins are modulated by two regulatory proteins: Sdc25 and Cdc25 GEFs. The cAMP/ PKA pathway helps in controlling the progression of the cell cycle at G1/ G0 stage. It also modulates the required cell size for mitosis and budding (Jian, 2010). The cAMP levels increase rapidly when starved yeast is fed with glucose or is subjected to intracellular acidification. The adenylate cyclase aids in cAMP synthesis, which occurs when the GTPases Ras components (Ras 1 and Ras 2) are activated by Guanine nucleotide exchange factors (Sdc25 and Cdc25) and GTPase- activating proteins (GAPs) such as Ira1p and Ira2p. The Gpa2 (heterotrimeric GTPase subunit) and Gpr1 (cognate receptor) modulate the activity of adenylate cyclase upon addition of glucose (Paiardi, 2007).
The Cdc25 and the Ras proteins play a critical role in the cAMP synthesis, cell viability and activity of the basal adenylate cyclase. The Ras has been shown to regulate the cAMP- PKA pathway, which is important for several cellular processes such as cell proliferation regulation, nutrient sensing, stress resistance and carbon storage. They usually localize themselves on the plasma membrane’s cytoplasmic face and interact with different cellular effectors to regulate cellular processes. The GTPase activating proteins negatively regulate Ras proteins while the Ras- GEFs positively regulates them. The Cdc25 has been shown to be crucial for activation of Ras2 and PKA pathways when CYR1 stimulates the adenylate cyclase that catalyzes the cAMP (Jones, 1991). The activated cAMP further activates the PKA pathways upon binding to the regulatory subunits and releasing the active catalytic subunits. However, the mechanisms and the signals leading to the Cdc25 activation are still unknown.
Role of Cdc25
For Ras, the Cdc25 protein is an exchange factor, which is essential for growth of Saccharomyces cerevisiae cells. The product of the Cdc25 gene is an approximately 180 kDa protein, which comprises of the domain that aids in exchange of guanine nucleotide activity in the C- terminal region. The gene product of Cdc25 contains the Ras- GEF, while the SDS25 is dispensable. It is expressed only in the presence of non- fermentable carbon source and during nutrient depletion (Vanoni, 1990). The important exchange domain is constrained up to 300- amino acids and it might have regulatory regions (Kaplon, 1995). The protein is highly unstable with a cyclin destruction box and its cellular contents act as a potentially limiting factor for its regulation. The regulation cdc25 for stability and adenylate cyclase interaction is highly dependent upon the large N- terminus. This also wields an inhibitory effect on the Cdc25 catalytic activity (Kaplon, 1995).
Role of Vid30c
The Vid30c is an essential GID complex as it facilitates degradation and ubiquitination of Mdh2p and FBPase. The complex is also important as it helps the cells to adapt to the gluconeogenic growth conditions once the glucose concentration increases. When glucose is abundant, the vid30c triggers Hxt3 expression and localizes the Hxt3p to the plasma membrane (Snowdon, 2008). However, when alcohol is the only carbon source that is present, the transcription of Hxt3 gets repressed. This results in endocytosis and degradation of the protein. This adaptation of yeast during the availability of different carbons sources is dependent on Vid30c (i.e., the core component: Vid24p, Vid30p; regulatory protein: Vid28p; catalytic component: Gid9p and Gid2p) (Snowdon, 2012). This is because in absence of these genes, the Hxt3p turnover and the repression of transcription get delayed (Snowdon, 2012). The Vid20c components are also essential for Hxt7p degradation induced by nitrogen starvation. Therefore, Vid30c is not only required for glucose replenishment but also is essential for aiding the organism to adapt to diverse nutrient conditions. Due to the predominant Hxt3p-GFP retention in the plasma membrane, the Ras/cAMP/PKA pathway prevents the Hxt3p turnover in the presence of ethanol especially when the cells lack BCY1 or express RASVAL19. Soulard (2010) supported this by proving that TORC1 inhibits the Ras/cAMP/PKA pathway activity when is treated with rapamycin.
Findings on vid30c participation in Cdc25 degradation in yeasts
Role of Cdc25 in glucose- induced Ras2 activation
Paiardi (2007) explained that Cdc25 protein is an important factor as it is essential for proliferation of yeast cells. However, they could not prove the exact mechanism using which the activity of Ras- GEF is regulated or how the nutrient gradient is transduced. The mechanism that triggers the complex to alter the Cdc25/Ras/cAMP pathway was also unknown. Earlier studies indicated that the regulator regions are present in the large protein. Thus, they investigated the mechanism behind the putative Cdc25 regulatory domains that acts using the nutrient sensing mechanism. Paiardi (2007) found that the Cdc25 is essential for the growth, which is substituted by the activity of the RasGEF . In their experiment, none of mutants created by them could modulate the protein content or the cell size when exposed to different nutrient environment. This indicated that the N- terminal region of Cdc25 plays a regulatory role and triggers the PKA activity, which is essential for the usual nutrient- sensing mechanism. The authors proved that when the CDC25 gene is replaced with either a homologous or a heterologous functional GEF domain, unregulated RasGEF activity permits a normal response to the glucose-induced cAMP. This is usually highly reliant on the Gpr1/Gpa2 signaling pathway. They further proved that Cdc25 might not be totally involved in the intracellular nutrient- sensing system.
Role of GID complex in carbohydrate metabolism
Santt (2008) prior to this study had reported that the gluconeogenesis to glycolysis switch is dependent upon the ubiquitin- proteasome that is responsible for the elimination of the fructose-1, 6-bisphosphate . The growth of yeast on the non- fermentable carbon sources is only possible in the presence of the FBPase. Therefore, in the presence of fermentable carbon source, the expression of FBP1 gene gets repressed and the enzyme as a result of allosteric inhibition is inactivated prior to degradation. Thus, the authors in their study, tried to characterize the Gid2/ Rmd5 contribution in FBPase proteasomal degradation. They also investigated whether the PEPCK degradation is influenced by Gid2/Rmd5. In their study, they showed in a genomic screen that degradation deficient (Gid)-proteins were induced by 7 glucose as a resultant of a complex formation, which binds to FBPase. Additionally, they found that Gid2/Rmd5 comprises of a disintegrated RING finger domain. Their results show that for FBPase degradation, the yeast cells might be using the pre-existing machinery in conditions that favours the autophagic process. However, in the presence of non- fermentable carbon source, the authors found that the proteasome triggers the FBPase degradation (Santt, 2008). It was learned from the genome- wide screen that, there are approximately 9 genes, whose deletions can result in impairment of the degradation of proteasomal- dependent FBPase when the cells are exposed to glucose enriched medium. They reported the few genes that were responsible for the degradation of vacuole- dependent FBPase are Gid1/Vid30, Gid5/Vid28, and Gid4/Vid24. The GID3 was found to encode for Ubc8 while GID6 genes were found to encode for Ubp14. The Gid2/ Rmd5 were found to belong to the 600 kDa soluble complex (such as Gid1/Vid30, Gid5/Vid28, Gid4/Vid24, Gid8, Gid9/Fyv10 and Gid7) (Ho, 2002). Santt (2008) also reported that in ethanol growing cells did not consist of Gid4/ Vid24 thereby, indicating that Gid4/ Vid24 is responsible the activation of Gid complex. In Gid4/Vid24 absence, the Gid7 or the Gid1/Vid30 remains in the complex form indicating that the Gid protein has no impact on the assemblage of the monomeric Gid proteins to the complex with higher molecular mass. Furthermore, the deletion of the Gid2/Rmd5 can also inhibit the FBPase polyubiquitination. The PEPCK was also shown to aid catabolite degradation of different gluconeogenic enzymes (Yin, 2000).
Role of Vid30c in Hxt turnover
Snowdon (2012) investigated the relation between the hexose transporter (Hxt) turnover and Vid30c. They reported that HXT3 transcription is repressed in the absence of glucose. This results in endocytosis and degradation of Hxt3 in a vacuole. The authors proved that during glucose starvation, the Vid30c plays a major role in Htx3 turnover. Additionally, the Vid30c has been reported to actively regulate the localization of Cdc25p and its stability (Jones, 1991). Snowdon (2012) showed that in rich nutrient conditions, the TORC1 pathway gets activated and aids in the progression of cell cycle. In glucose depleted state, the Snf1 pathway gets activated and exposes the cells to the gluconeogenic growth conditions (Jian, 2010). The authors in their manipulated the nitrogen and glucose signaling genes and delineated their role in regulatory events that is essential for Hxt3 and Hxt7 degradation. They found that the Snf1 has no role in this process. However, the endocytosis and amino acid permeases degradation was shown to be triggered by the Npr1p and TORC1 in a nitrogen- dependent manner and confirmed that rapamycin- induced Hxt7 degradation depends on Tor1 but not on Npr1 (Neklesa, 2009).
They showed that PKA prevents the Hxt3 and Hxt7 turnover. They supported their hypothesis by showing the predominant Hxt3p-GFP retention in the plasma membrane in the presence of ethanol when the cells lacked BCY1 or expressed RASVAL19 (Thevelein, 1999). Additionally, in nutrient rich or stress- free condition, they proved that PKA and TORC1 inhibits the cell cycle and inactivates Rim15. They showed that Rim15p helps in Hxt3p turnover in ethanol and Hxt7 upon rapamycin treatment. Thereby, proving that the Rim15 play an important role in the turnover of Hxt3 and Hxt7. It was also seen that during glucose starvation, the Hxt3 endocytosis is highly dependent on the Art8 and Rsp5, thereby proving that they play a pivotal role in Hxt3 endocytosis (Lin, 2008). However, during degradation of Hxt7, only Rsp5 is needed indicating that Art8 is highly specific to certain Hxt’s. The Vid40c and Rsp5 (E3 ubiquitin ligases) were also founded to be related to the Hxt3 and Hxt7 degradation where Rsp5 mediated ubiquitylation directly targets nutrient transporters while Vid30c does it indirectly .
Localization of Cdc25 RasGEF is PKA- dependent
Belotti (2011) proved that Cdc25 and Ira1 accumulate in the cell nucleus while Ira2, adenylate cyclase and Ras2 don’t. Upon analyzing the Cdc25 sequence, they found that it consist of several nuclear localization signals (NLS), which are imported into the nucleus. Ira-1 was also found to possess a NLS sequence in its C- terminal region confirming that the Ira1 and Ira2 have different roles (Belotti, 2011; Tanaka, 1990). They proved that RasGAP and RasGEF play a significant role in Ras signaling in the nucleus, however, this needs to be further confirmed using valid tests to prove that they result in activating a different target than adenylate cyclase. It was hypothesized that probably the PKA by using the TPK-1 dependent switch on activity and localization of Cdc25, controls the RasGAP and RasGEF functioning. After the PKA activation, Belotti (2011) showed the involvement of different phosphorylation sites in the dissociation of Cdc25 from Ras. Furthermore, the hyper- phosphorylated variants of cdc25 get abolished in the nutrient starvation state. Belotti (2011) stated that serine residues might be involved in the regulation of PKA- dependent localization of Cdc25 (Belotti, 2011; Tanaka, 1991). The Cdc25 re-localization post- glucose starvation indicates a transient and a strong increase in the cAMP levels when glucose is made available to the cells. During this process, the Ras signaling complex functions on the cell’s plasma membrane. However, the type of signal triggering the process is still to be confirmed. Belotti (2011) also showed that the RasC318S does not get palmitoylated and this cannot bind to the plasma membrane. Although it aids normal mitotic growth but the in presence of glucose it fails to induce the increase in expression of cAMP.
Relationship between Cdc25p and Ras2- GTP
Dong (2011) conducted a study to assess whether localization of Ras2p is dependent on the PKA activity and whether its activity is down- regulated by the PKA. They reported that the intracellular Ras2p localization is regulated by the PKA and the activation- state of the protein does not influence it. They showed that in a PKA- omitted mutant, the indistinguishable membrane of Ras2p and active Ras2val119 localization occurs independently. This proves that the Ras2p– GTP binding state has no role in the alteration of the protein conformation leading to the re-localization of intracellular Ras2p. Upon glucose signaling, the hyper- phosphorylated Ras2-GEF Cdc25p relocalizes to the cytoplasm from the membrane. The PKA activity and the PKA- regulated Cdc25 phosphorylation does not influences the Ras2p and Cdc25p intracellular association . The outcomes of their study revealed that there is a strong association of protein exists in between the Cdc25p (positive regulator) and Ras2p. This further eliminates the likelihood that the PKA down- regulates the Cdc25 by minimizing accessibility to the Ras2p target. They showed that serine 214 residue was the preferred site for phosphorylation that aids in promiscuous phosphorylation of Ras2p and augmented PKA activity (Tabba, 2010). Additionally, the enhanced PKA activity can lead to an insignificant difference in the localization and the membranes. They also showed that activation of Cyr1p is highly dependent upon the localization of plasma membrane of Ras2p, which in turn can decrease the activation of Cyr1p, thereby decreasing the cAMP signaling during synthesis (Matsumoto, 1982). Dong (2011) also proved that the PKA- attenuated cells down- regulates the binding capacity of Ras2p- GTP. The Ras2- GTP activity was inhibited as a result of phosphorylation of Cdc25p mediated by an unknown mechanism. The outcomes of this study proved that a significant portion of the Cdc25p interacts with the GDP- bound form of the Ras proteins. This catalyzes the exchange of nucleotide and produce Ras proteins that are GDP- bound. The study highlighted the fact that the dependence of the stability of Cdc25p–Ras2-GTP complex is a preferred protein association in different conformation which is highly influenced by the modification of Cdc25p phosphorylation. Furthermore, the PKA activity highly regulates the Ras2p localization. This down- regulates the Ras2p activity and the protein association amid of Ras2- GTP and Cdc25p are solely due to a reduced activity of Ras2-GEF Cdc25p (Jian, 2010).
Topology of Gid Complex in degradation of gluconeogenic enzymes
Menssen (2012) hypothesized that the Gid ubiquitin ligase comprises a total of 7 subunits; however, the actual arrangement of these subunits is yet to be determined. They evaluated the interactions between different subunit motifs by creating mutant versions of Gid proteins. They reported that Gid subunits are the key components of the E3 ligase and the Gid1 and Gi8 interacts in the absence of other subunits and their interactions are dependent on the LisH domains. Furthermore, the Gid8 was not found to enhance the Gid7 binding to Gid1. This indicates the existence of independent interaction between the CTLH and LisH motifs (Braun, 2011). Gid5 was identified as the adaptor molecule for the Gid4 regulatory subunit and the attachment highly depends on the Gid8 and Gid9 presence. Gid2 and Gid9 were found to comprise of a disintegrated RING domain, which forms a heterodimer (Deshaies, 2009). The outcomes of this study prove that Gid9 is the one that establishes contact with the complex. Additionally, the authors indicated that the Gid1, Gid5 and Gid2 are responsible for rapamycin- induced internalization and degradation of high- affinity Hxt7 using the endocytic pathway. The main function of the Gid proteins is to facilitate FBPase degradation that is vacuolar- dependent. Therefore, Menssen (2012) could successfully develop an initial model to identify the E3 ubiquitin ligase topology.
Conclusion
In conclusion, this literature review summarizes about the Vid30 complex participation in the degradation of CDC25 in yeast and their role in Ras/ cAMP/ PKA pathway (Van Der Merwe, 2001). Several qualitative and quantitative studies related to the topic was carefully reviewed to gain in- depth knowledge on the Vid30 complex and its influence on the Cdc25 degradation in the different nutrient environment. Pairidi (2007) proved that the activity of the low Ras guanine nucleotide exchange factor when unregulated permits the cAMP signaling that is induced by glucose and is highly intervened by the Grp1/Gpa2 system. On the other hand, Santt (2008) proved that the new ubiquitin complex, the Gid complex, comprises of novel subunits. They reported that Vid30c is important for degradation and ubiquitination of the Msh2p and FBPase. These subunits aid in catabolite degradation of different gluconeogenic enzymes. Additionally, the Vid24/Gid4 was also reported to be a potent regulator of the ubiquitin ligase activity. The study published by developed an initial model that helped in understanding the topology of the Gid complex. The adaptation highly depends on the Vid30c, specifically on the core components- Vid28p and Vid30p, regulatory protein- Vid24p and catalytic components- Gid2p and Gid9p. Snowdon (2011) showed that Vid30c is important for nitrogen regulated expression of genes, however, several components of this complex not only helps yeasts in adapting to glucose replenishment but also aids in adapting to the different nutrient environment.
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