L3 - Hierarchy of Atg

Question 1

can Atg1 localize to PAS without Atg13?

Answer 1

no

Question 2

can Atg13 localize to PAS without Atg1?

Answer 2

yes

Question 3

can Atg13 localize to PAS without Atg17?

Answer 3

no

Question 4

can Atg1 localize to PAS without Atg17?

Answer 4

no

Question 5

what kind of enzyme is Atg1?

Answer 5

kinase

Question 6

describe distribution of the TOR signalling pathway

Answer 6

part of a signalling pathway that is evolutionarily conserved

Question 7

what is the function of the TOR signalling pathway ?

Answer 7

regulates nutrient dependent cell growth by controlling machinery responsible for protein synthesis and transcription

Question 8

TOR

Answer 8

large ser/thr protein kinase

Question 9

describe structure of TOR

Answer 9

two multi-subunit protein complexes

Question 10

rapamycin

Answer 10

immunosuppressant that inhibits TORC1 activity

Question 11

when are TOR complexes upregulated?

Answer 11

nutrient rich conditions, promote cell growth

Question 12

what was the original hypothesized link between TOR and autophagy?

Answer 12

because TOR pathway regulates many processes in nutrient-dependent processes, Ohsumi thought TOR pathway might also have role in regu initiation of autophagy

Question 13

how did Ohsumi prove that there was a link betw TOR pathway and autophagy

Answer 13

ALP assay on different yeast strains with addition or rapamycin or tor mutants

Question 14

what happens to autophagy when you add rapamycin?

Answer 14

upregulated autophagy

Question 15

what happens to autophagy when you mutate TOR genes 1 and 2 and make then non-functional?

Answer 15

upregulated autophagy

Question 16

how many TOR genes do yeast encode?

Answer 16

tor1 and tor2

Question 17

why can you genetically delete tor1 but not tor2?

Answer 17

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)

Question 18

what were the conclusions of the TOR and autophagy experiments?

Answer 18

1) autophagy activated when TOR downregulated

2) rapamycin can be used to induce autophag instead of starving cells

Question 19

why is Atg1 so special amongst the other core Atg proteins, especially in the context of TOR pathway?

Answer 19

it is the only kinase of the 18 core Atg proteins so seems likely that it is the link to the TOR pathway

Question 20

what was the main challenge in studying Atg1 back then?

Answer 20

the exact substrate for Atg1 was not known

Question 21

what was tested to see if it was a substrate for Atg1?

Answer 21

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

Question 22

how did they find the regulators of Atg1?

Answer 22

yeast 2-hybrid assay

Question 23

what were the findings of the yeast 2-hybrid assay with Atg1?

Answer 23

1) Atg13 binds to Atg1

2) Atg 13 binds to Atg17

Question 24

how was the kinase activity of Atg1 quantified?

Answer 24

amount of MBP? (intensity of band in WB in the kinase assay)

Question 25

are Atg13 and Atg17 needed for Atg1?

Answer 25

yes, kinase assay showed that Atg13/Atg17 mutants had no kinase activity of Atg1 when treated with rapamycin

Question 26

how did they know that Atg13 was phosphorylated?

Answer 26

noticed smearing when testing an Atg13 Ab

Question 27

describe bands for Atg13-Ab and its phosphorylation state

Answer 27

1) nutrient rich: smearing of Atg13 band | 2) starved: significantly reduced smearing

Question 28

what did they find out about the phosphorylation of Atg13?

Answer 28

Atg13 hyperphosphorylated in nutrient rich conditions but dephosphorylated when autophagy is activated