L3 - Hierarchy of Atg Flashcards
can Atg1 localize to PAS without Atg13?
no
can Atg13 localize to PAS without Atg1?
yes
can Atg13 localize to PAS without Atg17?
no
can Atg1 localize to PAS without Atg17?
no
what kind of enzyme is Atg1?
kinase
describe distribution of the TOR signalling pathway
part of a signalling pathway that is evolutionarily conserved
what is the function of the TOR signalling pathway ?
regulates nutrient dependent cell growth by controlling machinery responsible for protein synthesis and transcription
TOR
large ser/thr protein kinase
describe structure of TOR
two multi-subunit protein complexes
rapamycin
immunosuppressant that inhibits TORC1 activity
when are TOR complexes upregulated?
nutrient rich conditions, promote cell growth
what was the original hypothesized link between TOR and autophagy?
because TOR pathway regulates many processes in nutrient-dependent processes, Ohsumi thought TOR pathway might also have role in regu initiation of autophagy
how did Ohsumi prove that there was a link betw TOR pathway and autophagy
ALP assay on different yeast strains with addition or rapamycin or tor mutants
what happens to autophagy when you add rapamycin?
upregulated autophagy
what happens to autophagy when you mutate TOR genes 1 and 2 and make then non-functional?
upregulated autophagy
how many TOR genes do yeast encode?
tor1 and tor2
why can you genetically delete tor1 but not tor2?
tor1 not required for viability. tor2 is essential for viability so can’t just delete or the cell will die => need to generate a temperature sensitive strain that loses function at 37C)
what were the conclusions of the TOR and autophagy experiments?
1) autophagy activated when TOR downregulated
2) rapamycin can be used to induce autophag instead of starving cells
why is Atg1 so special amongst the other core Atg proteins, especially in the context of TOR pathway?
it is the only kinase of the 18 core Atg proteins so seems likely that it is the link to the TOR pathway
what was the main challenge in studying Atg1 back then?
the exact substrate for Atg1 was not known
what was tested to see if it was a substrate for Atg1?
MBP (myelin basic protein), which is a model substrate for other ser/thr kinases and later found to be useful for quantifying the kinase activity of Atg1
how did they find the regulators of Atg1?
yeast 2-hybrid assay
what were the findings of the yeast 2-hybrid assay with Atg1?
1) Atg13 binds to Atg1
2) Atg 13 binds to Atg17
how was the kinase activity of Atg1 quantified?
amount of MBP? (intensity of band in WB in the kinase assay)
are Atg13 and Atg17 needed for Atg1?
yes, kinase assay showed that Atg13/Atg17 mutants had no kinase activity of Atg1 when treated with rapamycin
how did they know that Atg13 was phosphorylated?
noticed smearing when testing an Atg13 Ab
describe bands for Atg13-Ab and its phosphorylation state
1) nutrient rich: smearing of Atg13 band
2) starved: significantly reduced smearing
what did they find out about the phosphorylation of Atg13?
Atg13 hyperphosphorylated in nutrient rich conditions but dephosphorylated when autophagy is activated
