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6.2 Cell signalling in mating budding yeast, The mating of S. cerevisiae …
6.2 Cell signalling in mating budding yeast
Use these examples to give an account of the common features of signal pathways
The receptors for Ste2 and Ste3 are GPCRs
GPCRs bind to a wide range of signals
Small biogenic amines like adrenalin and histamine
Peptides like glucagon and parathyroid hormone
Models predict 800 human genes for GPCRs
up to 1700 in mice due to specialisation for olfaction
Evolutionarily ancient, predates other surface receptors including receptor tyrosine kinases
GPCR structure
Part of a group called 7 helix transmembrane receptors, 7TM, or serpentine receptors
N-terminus is outside and C terminus is inside the cell
Loops connecting the helices are where the magic happens
Extracellular loops contribute to the ligand-binding domain
Intracellular loops form the GEF domain, which interacts with the alpha subunit of the trimeric G-protein
Trimeric G-protein
Binds guanine nucleotides. Alpha and gamma subunits have lipid attachments that extend into the cell membrane.
Interacts with GTP and GDP
Can diffuse freely around the plane of the membrane.
Activation of the GPCR
Ligand binds GPCR
GPCR conformational change on the intracellular side
GPCR interacts with G-protein GEF domain, causes G-protein-bound GDP to exchange for GTP
G-protein dissociates from the GPCR, the alpha subunit dissociates from the beta and gamma subunits
The betagamma subunit can now interact with downstream elements.
When the alpha subunit-bound GTP is hydrolysed, the subunits reassemble
If the receptor is still bound to a ligand, the cycle may recommence
Ga-s G-proteins
Commonly activates adenylyl cylase via the alpha subunit
Active adenylyl cylase produces cyclic AMP (cyclic adenosine monophosphate, cAMP) from ATP
cAMP is a messenger that controls many aspects of metabolism, such as releasing glucose from glycogen and, in the heart, increasing the force of contraction
Most of the time, but not exclusively, cAMP activates protein kinase A, a serine-threonine kinase.
Included among PKA's targets are phosphodiesterases that can degrade cAMP into inactive AMP. This negative feedback loop mitigates PKA activity
Ligands of Ga-s GPCRs include
Adrenalin
Glucagon
Parathyroid hormone
Luteinising hormone
Adrenocorticotropic hormone
A peptide toxin from Vibrio cholerae causes cholera
This toxin covalently modifies Ga-s
Ga-s subunits become fixed on active, causing continuous cAMP production
elevated cAMP causes intestinal epithelial cells to continuously pump water and salts into the gut
Ga-i G-proteins
Ga-i is 'inhibitory' to Gary's 'stimulatory'
Inhibits adenylyl cylase , decreasing cAMP
Example ligands:
Somatostatin
Adrenalin
Acetylcholine
Glutamate
Covalent modification of Ga-i causes whooping cough
Toxic peptide emerges from Bordetella pertussis, aka pertussis toxin
Pertussis toxin locks Ga-i subunits in the inactive state
Therefore there is no interaction with adenylyl cylase, so cyclic AMP levels increase
Cellular effects of pertussis toxin are thus the same as with cholera. However the diseases are different because the pathogens target different parts of the body
Ga-q G-proteins
Activate phospholipase C (PLC)
Active PLC hydrolyses phosphotildylinositol 4,5-biphosphate (PIP-2)
Hydrolysis results in: inositol 1,4,5-triphosphate (IP3) and diacyglycerol (DAG)
These are both messengers with respective downstream effects
Ip3 triggers the release of Ca2+ ions from the endoplasmic reticulum
Ca2+ and DAG can synergistically activate protein kinase C
Common ligands: Adrenalin, histamine, endothelin, acetylcholine, vasopressin
Ca2+ has multiple downstream targets for changes to cell activity
How are other MAP kinase pathways similar? Make a comparison
Mammalian MAP kinase pathways are named because their target effector (probably usually) is a mitogen-activated kinase protein.
The mating MAP kinase pathway in S. Cerevisiae does not bind a mitogen, instead cell cycling is arrested.
It's considered a MAP kinase pathway because Fus3 (fusion 3) has sequence homology with mammalian MAP kinases.
In budding yeast mating MAP kinase pathway, the betagamma subunit dimer may be considered as both an anchor and a relay.
Also, Ste2 and Ste3 may be considered amplifiers because they can lead to the activation of many betagamma subunit dimers
Budding yeast has 5 discrete MAP kinase pathways
The other 4 are purposed for stress responses
Nitrogen deprivation
Activates the MAP kinase Kss1 which
Induces filamentous and invasive growth
High osmolarity
Activates the MAP kinase Hog1 which
Induces glycerol synthesis
Cell wall stress
Activates the MAP kinase Mpk1 which
Initiates cell wall repair
Carbon/nitrogen deprivation
Activates the MAP kinase Smk1 which
Induces sporulation
In 3 of the 5, Ste11 is phosphorylated by Ste20
The Kss1 pathway makes the cells of S. cerevisiae grow in an arranged, filamentous fashion. This helps the colony reach new nutrients.
The Kss1 pathway utilises elements of the Fus3 pathway, but their responses are mutually exclusive IE. Mating never occurs concurrently to filamentous growth and vice versa.
Key differences between the Fus3 pathway and the Kss1 pathway
Kss1 does not halt the cell cycle. Kss1 does not bind Far1
Kss1 is not localised. Kss1 does not use a scaffold such as Ste5. Growth in a particular direction is undesirable during filamentous growth.
There are no known feedback loops with Kss1, a sustained response is required.
If Ste12 is used in both the Fus3 and Kss1 pathways, how then does it elicit different gene expression?
When Ste12 is activated by Kss1, it also forms a heterodimer with another TF, Tec1
Activated Ste12 binds to the sequence TGAAACA, aka pheromone responsive element, PRE
Tec1 binds to the sequence CATTCTT, aka Tec1 consensus sequence, TCS
Ste12-Tec1 heterodimer binds to promoters containing both PRE and TCS, or just TCS.
Such promoters are found in genes required for filamentous growth.
When activated by Fus3, Ste12 forms Ste12-Ste12 homodimers
These homodimers bind to PRE pairs.
Such promoters are found in genes for mating
Fus3 also phosphorylates Tec1 to prevent filamentous growth
Cooperation between signal pathways
The Kss1 MAP kinase pathway can only progress if a cAMP-PKA pathway is also active
This is because yeast growth requires a fermentable carbon source
For carbon and energy, budding yeast has receptors that detect glucose in the environment.
The signal pathway for Gpr1 is reminiscent of the mammalian adenylyl cylase pathway, with Tpk homologous to protein kinase A.
The ACTUAL MAP kinase pathway of S. cerevisiae
Ste2 and Ste3 are the ligands
In this pathway, the active betagamma subunit transmits the signal
Ste20 is an enzyme, Ste5 is a scaffolding protein
Apart from the ste2 and Ste3 receptors, the pathway is identical between the two versions
Binding of the ligand causes dissociation of the G-protein from the GPCR
Betagamma subunit binds to an adapter protein called Far1
Far1 recruits another protein called CDC24 to the cell membrane
CDC24 is a member of a family of cell division cycle proteins
CDC24 is a guanine-nucleotide exchange factor (GEF), meaning it promotes the exchange of GDP for GTP on its target protein
CDC24 activates CDC42, a monomeric G-protein
Due to a high affinity for the betagamma subunit dimer, CDC42 is recruited to the membrane that will form the shmoo tip, as CDC42 organises actin filaments to promote contact with the other cell
Additionally, GTP-CDC42 binds and activates protein kinase Ste20
Simultaneously, the betagamma dimer also binds the scaffolding protein ste5
Ste5 binds st11, Ste7 and fus3
So...
A major role in the beta-gamma subunit dimer is to promote the formation of two complexes:
Far1, CDC24, CDC42, and Ste20
Ste5, Ste11, Ste7 and Fus3
Connecting them, ste11 is a substrate of phosphorylation by Ste20
This activates Ste11's own kinase activity
Phosphorylation cascades from Ste11 to Ste7 to MAP kinase fus3
Active Fus3 is released from Ste5
Fus3 diffuses throughout the cell, encountering and phosphorylating many proteins
Activated Fus3 can enter the nucleus and phosphorylate TF ste12, and also phosphorylate dig1 and dig2
Dig1 and dig2 normally prevent inactive ste12 from binding to DNA by holding it in a complex
Active Ste12 is free to bind to DNA and promote transcription of genes required for mating
Fus3 eventually is dephosphorylated by phosphatases, at which point it leaves the nucleus
Fus3 activation/deacttivation
Fus3 cannot be phosphorylated by Ste7 until it is bound by Ste5
It is believed that Ste5 alters the conformation of Fus3
Ste7 phosphorylates threonine 180 and tyrosine 182 on Fus3. It is thus known as a dual-specificity kinase
If the (subsequent) translocation of Fus3 to the nucleus is inhibited, the mating response does not occur.
The phosphate groups on T180 and Y182 are removed by phosphatases, deactivating Fus3
Redistribution of components of the Fus3 MAP kinase pathway
Maeder et al (2007) investigated the cellular distribution of Ste5, ste7 and Fus3 in the presence of pheromone.
Proteins were dyed green or red, and results were compared between untreated control cells or cells treated with 10 micrograms per ml alpha factor, for a couple of hours.
Conclusions
Unstimulated Ste7 is excluded from the nucleus, Ste5 and Fus3 are distributed between the nucleus and the cytosol.
In pheromone-stimulated cells each of these proteins localises at the shmoo-tip, with the effect less pronounced in Fus3
Regulation of budding yeast pheromone signaling
The influence of one signal pathway upon another is termed 'cross talk'
Fus3 mediates feedback by phosphorylating components of its own pathway.
Fus3 phosphorylates Ste11 and Far1
Ste11 phosphorylated by Fus3 gets marked for degradation
This is due to ubiquitination
Ubiquitin covalently added to a lysine aa residue
More ubiquitin is added to the first to form a chain
The proteasome recognises the ubiquitin, which then degrades the protein
Phosphorylation of Far1 by Fus3 halts the progression of the cell cycle
Phosphorylated Far1 moves to the nucleus
There, it binds with cycling, whose normal role is to proceed the cell cycle
Fus3's action on Ste11 and Far1 is negative feedback, because it terminates the MAP kinase pathway
Fus3 also enacts positive feedback
For example by phosphorylating Ste5, beleived to concentrate Ste5 near the GPCR.
TF Ste12 promotes Fus3 expression, a feedforward loop
The mating of S. cerevisiae
A colony contains both halploid and diploid cells
Diploid cells can reproduce 'vegetatively' by mitosis
Or they can mate to form a diploid zygote
Diploid cells replicate by mitosis
Haploid cells may perish umder starvation conditions
Diploid cells are able to produce spores that can withstand extreme environments
For mating to occur, haploids must change shape, come into contact and allow their membranes to fused
Haploids are either a or alpha
As determined by an allele at the MAT gene locus
These alleles encode TFs that drive expression of different pheromones
Pheromes disperse and can reach other haploids (diploids don't react to them)
The factor of one type only affects the cell of the other type because they have different receptors
a cells expresses Ste2 to receive alpha-factor
Alpha cells express Ste3 to receive a-factor
Signal reception activates the MAP kinase pathway
The first response is an alteration to the cytoskeleton that causes a protruding shmoo. This reaches towards the pheromone producer.