ethan Flashcards
what are different methods cells use to respond to stress in general
- perceive the threat
- acclimation: adjustment of gene expression: heat shock proteins, dehydrins, antifreeze proteins, membrane proteins, compatible solutes
- adaption: genetic differences mean better survival. evolution of the ability to acclimate
- apoptosis
- modification/repair of structures
- production of protective substances so stress doesnt occur in the first place
discuss principles of osmolarity
- glycerol (single ion): 0.05M glycerol will mean 0.05Mosmoles/litre
- potassium chloride (Kcl) (two ion); 0.05M of glycerol will mean 0.1 osmoles/per litre (twice the osmotic effect)
- high osmolality = high water potential
discuss the cellular response to osmotic stress
- something must be done because desiccating stresses (water potential outside cells is lower than inside) causes water to leave cells
- desiccating stress can occur due to drought, salt or freezing conditions
- solute accumulation inside cell decreases water potential (molecules get a shell of hydration) so no water flows out of the cell
- known as a “colligitive effect” (depending on the concnetraiton of the particle)
- a cell with 0.05M KCL (0.1 osmoles/l) will lose water when placed in a 0.4 osmoles/l environment
- a cell with 0.05KCL and glycerol accumulated 0.3M Glyceorl/l (total of 0.4osmoles/l) will not lose any water across its membrane because its already equilibrated with its environment
- other molecules and proteins are accumulated which have non-colligative effects (dont depend on concentration); antifreeze proteins alter water structure and inhibit freezing. other non-colligative molecules include sugars like sucrose.
- cells could alternatively take up solutes to reduce the water potential but these would have to be taken up in high concentrations and wouldn’t be compatible with normal cellular functioning so have not evolved to do this and instead synthesis their own compatible solutes
how does yeast respond to osmotic stress
- S. cerevisiae (baking and brewing) is often in environments of high salt/sugar concentration so has mechanisms of responding to osmotic stress
- 600 genes undergo major gene expression changes
- compatible solute accumulation (glycerol and trehalose)
describe glycerol synthesis
-HOG pathway signalling
-mutagenesis of HOG1 and PBS2 meant gene expression changes did not occur and the cells couldn’t survive osmotic stress)
-genes were sequenced and shown to be protein kinases
seqenced using phenotypic complementation (whole genome sequence not yet available); genome lysed into fragments using restriction enzymes and placing fragments in vectors which are introduced to yeast; yeast who can withstand stress must have HOG1/PSB2 gene so were sequenced
-when osmotic stress occurs, PSB2 phosphorylates HOG1 which moves into the nucleus and activates transcription factors which activate GPD1 and GPP2 expression; movement imaged using GFP. antibody binding showed that HOG1 does not increase in concentration but becomes phosphorylated
-GPD1 is expressed as glycerol-3-phosphate-dehydrogenase (glyceraldehyde-3-phosphate from glycolysis to glycerol-3-phosphate)
-GPP2 is expressed as glycerol-3-phosphatase (glycerol-3-phosphate to glycerol)
discuss MAPK cascades
-exist to amplify signals
-multiple cascades exist which can be connected or independant
-
discuss HOG pathway activators
- SLN1 inactivates HOG pathway under los osmotic stress
- those with mutated SLN1 die under low osmotic stress undless HOG1/PSB2 is mutated too in which case cells survive in low osmotic stress but not high osmotic stress.
- SLN1 mutants due because glycerol accumulation occur when it isn’t needed to water enters cells
- SLN1 is part of a two component sensor mechanism
- phenotypic complementation showed that it is a “histidine kinase sensor” protein which received an input and will likely have a “response regulator” which is the output module leadint to cellular change
- SSK1 is involved in HOG activation but isint the response regulator
- cells with mutated SLN1 genes may not die in low low osmotic stress if they have SSK1
- A two hybrid test showed that SLN1 and SSK1 dont interact
- YPD1 interacts with both SLN1 and SSK1 as part of a multi step two component system (3 proteins rather than 2)
- YPD1 mutants die under low osmotic stress but survive if HOG1/PSB2 is mutated too
- HOG1/PSB2 knockout causes death so the proteins must act in a chain type way
- phenotypic complementation suggested that the protein is homologous to the “histidine phosphorylation site” of the sensor protein
- two hybrid test showed interaction with both SLN1 and SSK1
- yeast have a more complicated two component system than usual bacteria
-under stressful conditions, SLN1 (inside membrane) detects the turgor pressure which signal osmolarity which causes a phosphorylation relay all the way to SSK1 which inactivates/deactivates HOG1
- Another signalling pathway exists involving SHO1
- those with mutated SSK2/SSK22 can withstand osmotic stress
- SHO1 and SSK22 mutation leads to death in high osmotic conditions
how are cell walls maintained during stressful conditions (low osmotic conditions)
-YPD1 phosphorylates and activates SKN7 (TF of genes needed to maintain cell walls)
what are the similarities with how eukaryotes deal with osmotic stress
- two component system exists in all animals apart from animals
- heterologous systems exist; A. thaliana ATHK1 gene was trasnferred to yeast without SLN1 and they could then survive
- ATHK1 does the opposite of SLN1 in plants; positive regulator which is needed for drought stress
- mammals have MAPK systems
- P38 is homologous to HOG1, and can be introduced into yeast
- eukaryotic cascade is more complicated than in bacteria
what makes up a usualy two component system
- histidine kinase sensor proteins
- (input and transmitter)
- percieve environmental signal at an input moduel and transmit the signal to its transmitter module which phosphorylates itself
- response regulator
- (reciever module and output)
- reciever module is phosphorylated by transmitter module, which causes the output module to have a conformational change and cellular change
how does the two-hybrid test work
- the beta-galactosidase reporter gene is activated by Gal4 enzyme
- gal4 consists of an activation domain and a DNA binding domain
- activation domain and DNA binding domain are seperated (SLN1 and SSK1),
- if the two components interact then the transcription activator will come together and express the reporter gene; blue colour
