Ras

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Ras is a switch

148-RasProtein_Ras.jpg Bild: Protein Data Base (hohe Auflösung)

  • membrane-bound receptors can switch it
  • GEFs switch it on
  • GAPs switch it off

Ras localizes to different membranes

Paper.svg Belotti (2012)
Localization of Ras signaling complex in budding yeast
PubMed (title) PubMed (ID) Google Vorlage:Paper
Paper.svg Bhattacharya (1995)
Ras membrane targeting is essential for glucose signaling but not for viability in yeast
PubMed (title) PubMed (ID) Google Vorlage:Paper
  • Ras cycling between PM, ER and Golgi (in dog kidney), in a manner regulated by palmitoylation (Lorentzen 2010)
  • ER, Golgi, PM, ... Mitos?
  • localization-dependent function?

Ras ist post-translationally modified: depending on it's localization/function ?

  • methylated
  • palmitoylated
  • farnesylated (simulataneous with palmitoylation? upon activation?)

Ras at the mitochondria may detect defective mitochondria via pH lowering

CO2 is an acid, that diffuses across membranes

CO2 is an acid: When dissolved in water, it forms the deprotonation equilibrium H2CO3 / HCO^3- / CO3^2-. This way, dissvoled CO2 leads to a lowering of pH. Additionally, CO2 is non-polar and thus capable of passing lipid bilayers. So, whereever it is produced inside the cell, it diffuses into all compartments and into the medium. If the medium is open to the air, CO2 can evaporate.

respiration deficiency lowers the pH

Glycolysis produces pyruvate. In the absence of oxygen the latter is transformed into ethanol and CO2, a lowering of pH takes place. In the presence of oxygen, mitochondrial metabolism takes places: Organic acid cycle (TCA): oxidation of carbon to CO2, and respiratory chain (RC): reduction of oxygen to water. These two processes are coupled: An "electron transfer" from TCA (FADH2 and NADH production) to RC (uptake of FADH2 and NADH) takes place. Simultaneously a "proton transfer" takes place: While TCA produces the acid CO2, which sets free protons, RC consumes protons to form water out of oxygen.

When oxidation and reduction processes function equally, pH stays constant.

Conclusively, in the presence of oxygen, a lowering of pH can be interpreted as an increased ratio of TCA / RC activity.

Questions:

  • If there happen to be superfluous protons, where are the electrons ?

They are on the reactive species, that leake from the respiratory chain (ROS).

  • How do the protons overcome the mitochondrial outer membrane, in order to be sensed by Ira1/2 (Ras) ?

unresolved

nitrogen starvation lowers the pH

Ras responds to pH lowering

Ras has been shown to respond to acidification (Colombo 1998). It changes it's influence on the adenylate cyclase Cyr1, which produces cAMP. cAMP activates the protein kinase A (PKA).

Addition of glucose leads to a transient cAMP response in the wild-type. This response is not conferred by Ras, but by Gpa2, which activates Cyr1 temporarily.

The constitutively active form of Ras (the 19:Val mutant), suppresses this cAMP response.

Question:

  • why? how can this be explained ?
  • Does Ras bind GTP or GDP upon acidification ?
  • Is Ras-GTP activating or inhibiting the adenylate cyclase ?

GTP, leaking from mitochondria, may stimulate Ras activation

Ras's activating substrate is GTP. GTP is also a product of the organic acid cycle - GTP, a signalling molecule, rather than ATP, the chemically homologous energy equivalent. It is usually argued, that GTP is produced to gain energy. For no obvious reason, still, the production of GTP is conserved over the production of ATP, indicating that GTP at least fulfills a second function. This idea is supported by the fact, that a GTP-ATP-transforming enzyme has evolved, although the SuccinylCoA-synthetase could presumably have much "easier" evolved to producing ATP instead of GTP.

It is noteworthy, that GTP production takes place, after ketoglutarate production, which may support one of my previous hypothesises, in which the degradation of ketoglutarate must be inhibited under nitrogen starvation to enable efficient ammonia assimilation.

In any case, I suspect, that Ras signalling may be controlled two-fold at the mitochondria. However, I am not aware of a mechanism transporting GTP across the mitochondrial membrane. If there is none, mitochondrial membrane disruption would be the only circumstance, leading to mitochondrial GTP binding Ras, which would still make perfect sense in the context of the Ras - mitophagy hypothesis.

Question:

  • how could GTP pass the mitochondrial membrane?
  • where is the GTP-ATP-transformation taking place?
    • is GTP transported across the mitochondrial membrane?

Conclusion

Ras responds to acidification, which occurs upon respiratory chain deficiency, so Ras actually "measures" the respiration efficiency, respectively the "mitochondrial quality". It is therefore probable, that Ras is also involved in mitophagy. However, no experiments have been carried out addressing this hypothesis so far.

Questions:

  • How is Ras localized, specifically to the mitochondria?

unresolved. Palmitoylation has been shown to confer membrane affinity, but doesn't explain special membrane localizations. Another "signal" or transport is expected.

  • Is this localization required for mitophagy?
  • How is the mitochondrially localized Ras inactivated in the absence of oxygen?

Ras is switched by Cdc25, Sdc25, Ira1 and Ira2

very interesting

  • Ira1/2: sense pH lowering!

My Ras hypothesis could beconfirmed, if there was experimental evidence, that a special, modifed Ras and/or Ira1 and/or Ira2 specifically localize to the mitochondria.

more stuff

properties

This indicates, that stress induction is due to the absence of Ras activity, which requires GTP being bound.

Ras is localized to the mitochondria in whi2 mutants

Müller, Reichert

No direct interaction between Ras and Whi2 has been found, so since Whi2 is required for Msn induced stress response, it is possible, that a stress response is responsible for Ras degradation.

Ras and Cyr1 at the mitochondria ?

Ras recruits Tsa1 and Cyr1 to the mitochondria. The latter actually makes sense, since ATP, the substrate of Cyr1, is -in the presence of oxygen- mainly produced by mitochondrial metabolism. ... Therefore absence of Ras could lead to reduced cAMP levels due to improper localization of Cyr1, and thus to downregulation of PKA signalling. Improper downregulation of PKA signalling in turn could explain induction of stress response.

If this is true, the Ras degradation mechanism could be mitophagy.

Ras's database interactions

Protein interaction analyses does not include "interesting" proteins, only Palmitolytransferase, the GEF Cdc25, the GEF Lte1, Hsp82, Thioredoxinperoxidase Tsa1 which confers resistance to oxidative stress, Calmodulin-dependent kinase Cmk1 (why?) and G2/M transition kinase Swe1.

Mammalian Ras

  • Ras isoforms H-Ras and N-Ras ?
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